Compositions and methods for modulating complement factor B expression

ABSTRACT

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway by administering a Complement Factor B (CFB) specific inhibitor to a subject.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0251USASEQ_ST25.txt created Oct. 26, 2016, which is 204 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway by administering a Complement Factor B (CFB) specific inhibitor to a subject.

BACKGROUND

The complement system is part of the host innate immune system involved in lysing foreign cells, enhancing phagocytosis of antigens, clumping antigen-bearing agents, and attracting macrophages and neutrophils. The complement system is divided into three initiation pathways—the classical, lectin, and alternative pathways—that converge at component C3 to generate an enzyme complex known as C3 convertase, which cleaves C3 into C3a and C3b. C3b associates with C3 convertase mediated by CFB and results in generation of C5 convertase, which cleaves C5 into C5a and C5b, which initiates the membrane attack pathway resulting in the formation of the membrane attack complex (MAC) comprising components C5b, C6, C7, C8, and C9. The membrane-attack complex (MAC) forms transmembrane channels and disrupts the phospholipid bilayer of target cells, leading to cell lysis.

In the homeostatic state, the alternative pathway is continuously activated at a low “tickover” level as a result of activation of the alternative pathway by spontaneous hydrolysis of C3 and the production of C3b, which generates C5 convertase.

SUMMARY

The complement system mediates innate immunity and plays an important role in normal inflammatory response to injury, but its dysregulation may cause severe injury. Activation of the alternative complement pathway beyond its constitutive “tickover” level can lead to unrestrained hyperactivity and manifest as diseases of complement dysregulation.

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject by administration of a Complement Factor B (CFB) specific inhibitor. Several embodiments provided herein are drawn to a method of inhibiting expression of CFB in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway by administering a CFB specific inhibitor to the subject. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a CFB specific inhibitor to the subject. In several embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a CFB specific inhibitor to the subject.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^(st) edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

“2′-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine in a 2′-F nucleoside is in the ribo position (replacing the OH of a natural ribose).

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxyethyl modification at the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanosyl ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications.

“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.

“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within +10% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of CFB”, it is implied that CFB levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

“Alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

“Bicyclic nucleic acid” or “BNA” or “BNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4′ and 2′position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, as depicted below.

As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R₁)(R₂)_(n)]—, —C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—, —S(═O)_(x)— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R₁ and R₂ is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: —[C(R₁)(R₂)_(n)]—, —[C(R₁)(R₂)_(n)]—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—. Furthermore, other bridging groups encompassed with the definition of LNA are 4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′,4′-CH₂—O-2′,4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′-bridges, wherein each R₁ and R₂ is, independently, H, a protecting group or C₁-C₁₂ alkyl.

Also included within the definition of LNA according to the invention are LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′ carbon atom of the sugar ring, thereby forming a methyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (—CH₂—) group connecting the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicylic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy (4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is also encompassed within the definition of LNA, as used herein.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“Carbohydrate” means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

“Carbohydrate cluster” means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

“Carbohydrate derivative” means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.

“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.

“Cleavable bond” means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.

“Cleavable moiety” means a bond or group that is capable of being split under physiological conditions. In certain embodiments, a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as a lysosome. In certain embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.

“Conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

“conjugate linker” or “linker” in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link (1) an oligonucleotide to another portion of the conjugate group or (2) two or more portions of the conjugate group.

Conjugate groups are shown herein as radicals, providing a bond for forming covalent attachment to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3′-oxygen atom of the 3′-hydroxyl group of the 3′ terminal nucleoside of the oligomeric compound. In certain embodiments the point of attachment on the oligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group of the 5′ terminal nucleoside of the oligomeric compound. In certain embodiments, the bond for forming attachment to the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster portion comprises: a targeting moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups and is designated “GalNAc3”. In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated “GalNAc4”. Specific carbohydrate cluster portions (having specific tether, branching and conjugate linker groups) are described herein and designated by Roman numeral followed by subscript “a”. Accordingly “GalNac3-1a” refers to a specific carbohydrate cluster portion of a conjugate group having 3 GalNac groups and specifically identified tether, branching and linking groups. Such carbohydrate cluster fragment is attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.

“Conjugate compound” means any atoms, group of atoms, or group of linked atoms suitable for use as a conjugate group. In certain embodiments, conjugate compounds may possess or impart one or more properties, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Complement Factor B (CFB)” means any nucleic acid or protein of CFB. “CFB nucleic acid” means any nucleic acid encoding CFB. For example, in certain embodiments, a CFB nucleic acid includes a DNA sequence encoding CFB, an RNA sequence transcribed from DNA encoding CFB (including genomic DNA comprising introns and exons), including a non-protein encoding (i.e. non-coding) RNA sequence, and an mRNA sequence encoding CFB. “CFB mRNA” means an mRNA encoding a CFB protein.

“CFB specific inhibitor” refers to any agent capable of specifically inhibiting CFB RNA and/or CFB protein expression or activity at the molecular level. For example, CFB specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of CFB RNA and/or CFB protein.

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

“Designing” or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

“Differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

“Double-stranded” refers to two separate oligomeric compounds that are hybridized to one another. Such double stranded compounds may have one or more or non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.

“Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”

“Halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

“Heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

“Identifying an animal having, or at risk for having, a disease, disorder and/or condition” means identifying an animal having been diagnosed with the disease, disorder and/or condition or identifying an animal predisposed to develop the disease, disorder and/or condition. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Internucleoside neutral linking group” means a neutral linking group that directly links two nucleosides.

“Internucleoside phosphorus linking group” means a phosphorus linking group that directly links two nucleosides.

“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.

“Linkage motif” means a pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′bridge.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety.

“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating CFB mRNA can mean to increase or decrease the level of CFB mRNA and/or CFB protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, a CFB antisense compound can be a modulator that decreases the amount of CFB mRNA and/or CFB protein in a cell, tissue, organ or organism.

“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Mono or polycyclic ring system” is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, hetero-aromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to hetero-cyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two ═O groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.

“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Neutral linking group” means a linking group that is not charged. Neutral linking groups include without limitation phospho-triesters, methylphosphonates, MMI (—CH2-N(CH3)-O—), amide-3 (—CH2-C(═O)—N(H)—), amide-4 (—CH2-N(H)—C(═O)—), formacetal (—O—CH2-O—), and thioformacetal (—S—CH2-O—). Further neutral linking groups include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)). Further neutral linking groups include nonionic linkages comprising mixed N, O, S and CH2 component parts.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Non-internucleoside neutral linking group” means a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral linking group links two groups, neither of which is a nucleoside.

“Non-internucleoside phosphorus linking group” means a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

“Nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleoside motif” means a pattern of nucleoside modifications in an oligonucleotide or a region thereof. The linkages of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

“Phosphorus linking group” means a linking group comprising a phosphorus atom. Phosphorus linking groups include without limitation groups having the formula:

wherein:

R_(a) and R_(d) are each, independently, O, S, CH₂, NH, or NJ₁ wherein J₁ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino; and

J₁ is R_(b) is O or S.

Phosphorus linking groups include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound

“Prevent” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.

“Prodrug” means an inactive or less active form of a compound which, when administered to a subject, is metabolized to form the active, or more active, compound (e.g., drug).

“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

“Protecting group” means any compound or protecting group known to those having skill in the art. Non-limiting examples of protecting groups may be found in “Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“RISC based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to the RNA Induced Silencing Complex (RISC).

“RNase H based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Separate regions” means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.

“Sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

“Side effects” means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Slows progression” means decrease in the development of the said disease.

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substuent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unpro-tected. In certain embodiments, compounds of the present disclosure have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydro-carbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms that differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C¬(O)¬Raa), carboxyl (—C(O)O-Raa), aliphatic groups, ali-cyclic groups, alkoxy, substituted oxy (—O—Raa), aryl, aralkyl, heterocyclic radical, hetero¬aryl, hetero-arylalkyl, amino (N(Rbb)¬(Rcc)), imino (═NRbb), amido (C(O)N¬(Rbb)(Rcc) or N(Rbb)C(O)Raa), azido (—N3), nitro (NO2), cyano (—CN), carbamido (OC(O)N(Rbb)(Rcc) or N(Rbb)¬C(O)—ORaa), ureido (N(Rbb)C(O)¬N(Rbb)(Rcc)), thioureido (N(Rbb)C ¬¬¬(S)N(Rbb)¬(Rcc)), guanidinyl (N(Rbb)¬C(═NRbb)¬N(Rbb)(Rcc)), amidinyl (C(═NRbb)¬¬N(Rbb)(Rcc) or N(Rbb)C(═NRbb)(Raa)), thiol (O—SRbb), sulfinyl (S(O)Rbb), sulfonyl (—S(O)₂Rbb) and sulfonamidyl (—S(O)₂N(Rbb)(Rcc) or N(Rbb)¬S¬¬(O)₂Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and hetero¬aryl¬alkyl. Selected substituents within the compounds described herein are present to a recursive degree.

“Substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.

“Sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

“Sugar motif” means a pattern of sugar modifications in an oligonucleotide or a region thereof.

“Sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.

“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

“Terminal internucleoside linkage” means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“Treat” refers to administering a pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal. In certain embodiments, one or more pharmaceutical compositions can be administered to the animal.

“Unmodified” nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

Certain embodiments provide methods, compounds and compositions for inhibiting Complement Factor B (CFB) expression.

Certain embodiments provide antisense compounds targeted to a CFB nucleic acid. In certain embodiments, the CFB nucleic acid has the sequence set forth in GENBANK Accession No. NM_001710.5 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No NW_001116486.1 truncated from nucleotides 536000 to 545000 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. XM_001113553.2 (incorporated herein as SEQ ID NO: 4), or GENBANK Accession No. NM_008198.2 (incorporated herein as SEQ ID NO: 5).

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 9 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 11 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 12 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleobases 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631 of SEQ ID NO: 1, and wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631 of SEQ ID NO:1, and wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleobases 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, or 7846-7862 of SEQ ID NO: 2, and wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862 of SEQ ID NO: 2, and wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

In certain embodiments, antisense compounds or oligonucleotides target a region of a CFB nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a CFB nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion of a region recited herein. In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting any of the following nucleotide regions of SEQ ID NO: 1: 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, and 2616-2631.

In certain embodiments, antisense compounds or oligonucleotides target a region of a CFB nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a CFB nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion of a region recited herein. In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting the following nucleotide regions of SEQ ID NO: 2: 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862.

In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting the 3′UTR of a CFB nucleic acid. In certain aspects, the modified oligonucleotide targets within nucleotides 2574-2626 of a CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1. In certain aspects, the modified oligonucleotide has at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion within nucleotides 2574-2626 of a CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1.

In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting a region of a CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1 within nucleobases 2457-2631, 2457-2472, 2457-2474, 2457-2476, 2457-2566, 2457-2570, 2457-2571, 2457-2572, 2457-2573, 2457-2574, 2457-2575, 2457-2576, 2457-2577, 2457-2578, 2457-2579, 2457-2580, 2457-2581, 2457-2582, 2457-2583, 2457-2584, 2457-2585, 2457-2586, 2457-2587, 2457-2588, 2457-2589, 2457-2590, 2457-2591, 2457-2592, 2457-2593, 2457-2594, 2457-2595, 2457-2596, 2457-2597, 2457-2598, 2457-2599, 2457-2600, 2457-2601, 2457-2602, 2457-2603, 2457-2604, 2457-2605, 2457-2606, 2457-2607, 2457-2608, 2457-2609, 2457-2610, 2457-2611, 2457-2612, 2457-2613, 2457-2614, 2457-2615, 2457-2616, 2457-2617, 2457-2618, 2457-2619, 2457-2620, 2457-2621, 2457-2622, 2457-2623, 2457-2624, 2457-2625, 2457-2626, 2457-2627, 2457-2628, 2457-2629, 2457-2630, 2457-2631, 2459-2474, 2459-2476, 2459-2566, 2459-2570, 2459-2571, 2459-2572, 2459-2573, 2459-2574, 2459-2575, 2459-2576, 2459-2577, 2459-2578, 2459-2579, 2459-2580, 2459-2581, 2459-2582, 2459-2583, 2459-2584, 2459-2585, 2459-2586, 2459-2587, 2459-2588, 2459-2589, 2459-2590, 2459-2591, 2459-2592, 2459-2593, 2459-2594, 2459-2595, 2459-2596, 2459-2597, 2459-2598, 2459-2599, 2459-2600, 2459-2601, 2459-2602, 2459-2603, 2459-2604, 2459-2605, 2459-2606, 2459-2607, 2459-2608, 2459-2609, 2459-2610, 2459-2611, 2459-2612, 2459-2613, 2459-2614, 2459-2615, 2459-2616, 2459-2617, 2459-2618, 2459-2619, 2459-2620, 2459-2621, 2459-2622, 2459-2623, 2459-2624, 2459-2625, 2459-2626, 2459-2627, 2459-2628, 2459-2629, 2459-2630, 2459-2631, 2461-2476, 2461-2566, 2461-2570, 2461-2571, 2461-2572, 2461-2573, 2461-2574, 2461-2575, 2461-2576, 2461-2577, 2461-2578, 2461-2579, 2461-2580, 2461-2581, 2461-2582, 2461-2583, 2461-2584, 2461-2585, 2461-2586, 2461-2587, 2461-2588, 2461-2589, 2461-2590, 2461-2591, 2461-2592, 2461-2593, 2461-2594, 2461-2595, 2461-2596, 2461-2597, 2461-2598, 2461-2599, 2461-2600, 2461-2601, 2461-2602, 2461-2603, 2461-2604, 2461-2605, 2461-2606, 2461-2607, 2461-2608, 2461-2609, 2461-2610, 2461-2611, 2461-2612, 2461-2613, 2461-2614, 2461-2615, 2461-2616, 2461-2617, 2461-2618, 2461-2619, 2461-2620, 2461-2621, 2461-2622, 2461-2623, 2461-2624, 2461-2625, 2461-2626, 2461-2627, 2461-2628, 2461-2629, 2461-2630, 2461-2631, 2551-2566, 2551-2570, 2551-2571, 2551-2572, 2551-2573, 2551-2574, 2551-2575, 2551-2576, 2551-2577, 2551-2578, 2551-2579, 2551-2580, 2551-2581, 2551-2582, 2551-2583, 2551-2584, 2551-2585, 2551-2586, 2551-2587, 2551-2588, 2551-2589, 2551-2590, 2551-2591, 2551-2592, 2551-2593, 2551-2594, 2551-2595, 2551-2596, 2551-2597, 2551-2598, 2551-2599, 2551-2600, 2551-2601, 2551-2602, 2551-2603, 2551-2604, 2551-2605, 2551-2606, 2551-2607, 2551-2608, 2551-2609, 2551-2610, 2551-2611, 2551-2612, 2551-2613, 2551-2614, 2551-2615, 2551-2616, 2551-2617, 2551-2618, 2551-2619, 2551-2620, 2551-2621, 2551-2622, 2551-2623, 2551-2624, 2551-2625, 2551-2626, 2551-2627, 2551-2628, 2551-2629, 2551-2630, 2551-2631, 2553-2570, 2553-2571, 2553-2572, 2553-2573, 2553-2574, 2553-2575, 2553-2576, 2553-2577, 2553-2578, 2553-2579, 2553-2580, 2553-2581, 2553-2582, 2553-2583, 2553-2584, 2553-2585, 2553-2586, 2553-2587, 2553-2588, 2553-2589, 2553-2590, 2553-2591, 2553-2592, 2553-2593, 2553-2594, 2553-2595, 2553-2596, 2553-2597, 2553-2598, 2553-2599, 2553-2600, 2553-2601, 2553-2602, 2553-2603, 2553-2604, 2553-2605, 2553-2606, 2553-2607, 2553-2608, 2553-2609, 2553-2610, 2553-2611, 2553-2612, 2553-2613, 2553-2614, 2553-2615, 2553-2616, 2553-2617, 2553-2618, 2553-2619, 2553-2620, 2553-2621, 2553-2622, 2553-2623, 2553-2624, 2553-2625, 2553-2626, 2553-2627, 2553-2628, 2553-2629, 2553-2630, 2553-2631, 2554-2573, 2554-2574, 2554-2575, 2554-2576, 2554-2577, 2554-2578, 2554-2579, 2554-2580, 2554-2581, 2554-2582, 2554-2583, 2554-2584, 2554-2585, 2554-2586, 2554-2587, 2554-2588, 2554-2589, 2554-2590, 2554-2591, 2554-2592, 2554-2593, 2554-2594, 2554-2595, 2554-2596, 2554-2597, 2554-2598, 2554-2599, 2554-2600, 2554-2601, 2554-2602, 2554-2603, 2554-2604, 2554-2605, 2554-2606, 2554-2607, 2554-2608, 2554-2609, 2554-2610, 2554-2611, 2554-2612, 2554-2613, 2554-2614, 2554-2615, 2554-2616, 2554-2617, 2554-2618, 2554-2619, 2554-2620, 2554-2621, 2554-2622, 2554-2623, 2554-2624, 2554-2625, 2554-2626, 2554-2627, 2554-2628, 2554-2629, 2554-2630, 2554-2631, 2555-2572, 2555-2573, 2555-2574, 2555-2575, 2555-2576, 2555-2577, 2555-2578, 2555-2579, 2555-2580, 2555-2581, 2555-2582, 2555-2583, 2555-2584, 2555-2585, 2555-2586, 2555-2587, 2555-2588, 2555-2589, 2555-2590, 2555-2591, 2555-2592, 2555-2593, 2555-2594, 2555-2595, 2555-2596, 2555-2597, 2555-2598, 2555-2599, 2555-2600, 2555-2601, 2555-2602, 2555-2603, 2555-2604, 2555-2605, 2555-2606, 2555-2607, 2555-2608, 2555-2609, 2555-2610, 2555-2611, 2555-2612, 2555-2613, 2555-2614, 2555-2615, 2555-2616, 2555-2617, 2555-2618, 2555-2619, 2555-2620, 2555-2621, 2555-2622, 2555-2623, 2555-2624, 2555-2625, 2555-2626, 2555-2627, 2555-2628, 2555-2629, 2555-2630, 2555-2631, 2556-2573, 2556-2574, 2556-2575, 2556-2576, 2556-2577, 2556-2578, 2556-2579, 2556-2580, 2556-2581, 2556-2582, 2556-2583, 2556-2584, 2556-2585, 2556-2586, 2556-2587, 2556-2588, 2556-2589, 2556-2590, 2556-2591, 2556-2592, 2556-2593, 2556-2594, 2556-2595, 2556-2596, 2556-2597, 2556-2598, 2556-2599, 2556-2600, 2556-2601, 2556-2602, 2556-2603, 2556-2604, 2556-2605, 2556-2606, 2556-2607, 2556-2608, 2556-2609, 2556-2610, 2556-2611, 2556-2612, 2556-2613, 2556-2614, 2556-2615, 2556-2616, 2556-2617, 2556-2618, 2556-2619, 2556-2620, 2556-2621, 2556-2622, 2556-2623, 2556-2624, 2556-2625, 2556-2626, 2556-2627, 2556-2628, 2556-2629, 2556-2630, 2556-2631, 2557-2574, 2557-2575, 2557-2576, 2557-2577, 2557-2578, 2557-2579, 2557-2580, 2557-2581, 2557-2582, 2557-2583, 2557-2584, 2557-2585, 2557-2586, 2557-2587, 2557-2588, 2557-2589, 2557-2590, 2557-2591, 2557-2592, 2557-2593, 2557-2594, 2557-2595, 2557-2596, 2557-2597, 2557-2598, 2557-2599, 2557-2600, 2557-2601, 2557-2602, 2557-2603, 2557-2604, 2557-2605, 2557-2606, 2557-2607, 2557-2608, 2557-2609, 2557-2610, 2557-2611, 2557-2612, 2557-2613, 2557-2614, 2557-2615, 2557-2616, 2557-2617, 2557-2618, 2557-2619, 2557-2620, 2557-2621, 2557-2622, 2557-2623, 2557-2624, 2557-2625, 2557-2626, 2557-2627, 2557-2628, 2557-2629, 2557-2630, 2557-2631, 2558-2575, 2558-2576, 2558-2577, 2558-2578, 2558-2579, 2558-2580, 2558-2581, 2558-2582, 2558-2583, 2558-2584, 2558-2585, 2558-2586, 2558-2587, 2558-2588, 2558-2589, 2558-2590, 2558-2591, 2558-2592, 2558-2593, 2558-2594, 2558-2595, 2558-2596, 2558-2597, 2558-2598, 2558-2599, 2558-2600, 2558-2601, 2558-2602, 2558-2603, 2558-2604, 2558-2605, 2558-2606, 2558-2607, 2558-2608, 2558-2609, 2558-2610, 2558-2611, 2558-2612, 2558-2613, 2558-2614, 2558-2615, 2558-2616, 2558-2617, 2558-2618, 2558-2619, 2558-2620, 2558-2621, 2558-2622, 2558-2623, 2558-2624, 2558-2625, 2558-2626, 2558-2627, 2558-2628, 2558-2629, 2558-2630, 2558-2631, 2559-2576, 2559-2577, 2559-2578, 2559-2579, 2559-2580, 2559-2581, 2559-2582, 2559-2583, 2559-2584, 2559-2585, 2559-2586, 2559-2587, 2559-2588, 2559-2589, 2559-2590, 2559-2591, 2559-2592, 2559-2593, 2559-2594, 2559-2595, 2559-2596, 2559-2597, 2559-2598, 2559-2599, 2559-2600, 2559-2601, 2559-2602, 2559-2603, 2559-2604, 2559-2605, 2559-2606, 2559-2607, 2559-2608, 2559-2609, 2559-2610, 2559-2611, 2559-2612, 2559-2613, 2559-2614, 2559-2615, 2559-2616, 2559-2617, 2559-2618, 2559-2619, 2559-2620, 2559-2621, 2559-2622, 2559-2623, 2559-2624, 2559-2625, 2559-2626, 2559-2627, 2559-2628, 2559-2629, 2559-2630, 2559-2631, 2560-2577, 2560-2578, 2560-2579, 2560-2580, 2560-2581, 2560-2582, 2560-2583, 2560-2584, 2560-2585, 2560-2586, 2560-2587, 2560-2588, 2560-2589, 2560-2590, 2560-2591, 2560-2592, 2560-2593, 2560-2594, 2560-2595, 2560-2596, 2560-2597, 2560-2598, 2560-2599, 2560-2600, 2560-2601, 2560-2602, 2560-2603, 2560-2604, 2560-2605, 2560-2606, 2560-2607, 2560-2608, 2560-2609, 2560-2610, 2560-2611, 2560-2612, 2560-2613, 2560-2614, 2560-2615, 2560-2616, 2560-2617, 2560-2618, 2560-2619, 2560-2620, 2560-2621, 2560-2622, 2560-2623, 2560-2624, 2560-2625, 2560-2626, 2560-2627, 2560-2628, 2560-2629, 2560-2630, 2560-2631, 2561-2578, 2561-2579, 2561-2580, 2561-2581, 2561-2582, 2561-2583, 2561-2584, 2561-2585, 2561-2586, 2561-2587, 2561-2588, 2561-2589, 2561-2590, 2561-2591, 2561-2592, 2561-2593, 2561-2594, 2561-2595, 2561-2596, 2561-2597, 2561-2598, 2561-2599, 2561-2600, 2561-2601, 2561-2602, 2561-2603, 2561-2604, 2561-2605, 2561-2606, 2561-2607, 2561-2608, 2561-2609, 2561-2610, 2561-2611, 2561-2612, 2561-2613, 2561-2614, 2561-2615, 2561-2616, 2561-2617, 2561-2618, 2561-2619, 2561-2620, 2561-2621, 2561-2622, 2561-2623, 2561-2624, 2561-2625, 2561-2626, 2561-2627, 2561-2628, 2561-2629, 2561-2630, 2561-2631, 2562-2577, 2562-2578, 2562-2579, 2562-2580, 2562-2581, 2562-2582, 2562-2583, 2562-2584, 2562-2585, 2562-2586, 2562-2587, 2562-2588, 2562-2589, 2562-2590, 2562-2591, 2562-2592, 2562-2593, 2562-2594, 2562-2595, 2562-2596, 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2591-2631, 2592-2611, 2592-2612, 2592-2613, 2592-2614, 2592-2615, 2592-2616, 2592-2617, 2592-2618, 2592-2619, 2592-2620, 2592-2621, 2592-2622, 2592-2623, 2592-2624, 2592-2625, 2592-2626, 2592-2627, 2592-2628, 2592-2629, 2592-2630, 2592-2631, 2593-2608, 2593-2612, 2593-2613, 2593-2614, 2593-2615, 2593-2616, 2593-2617, 2593-2618, 2593-2619, 2593-2620, 2593-2621, 2593-2622, 2593-2623, 2593-2624, 2593-2625, 2593-2626, 2593-2627, 2593-2628, 2593-2629, 2593-2630, 2593-2631, 2594-2612, 2594-2613, 2594-2614, 2594-2615, 2594-2616, 2594-2617, 2594-2618, 2594-2619, 2594-2620, 2594-2621, 2594-2622, 2594-2623, 2594-2624, 2594-2625, 2594-2626, 2594-2627, 2594-2628, 2594-2629, 2594-2630, 2594-2631, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2595-2615, 2595-2616, 2595-2617, 2595-2618, 2595-2619, 2595-2620, 2595-2621, 2595-2622, 2595-2623, 2595-2624, 2595-2625, 2595-2626, 2595-2627, 2595-2628, 2595-2629, 2595-2630, 2595-2631, 2596-2614, 2596-2615, 2596-2616, 2596-2617, 2596-2618, 2596-2619, 2596-2620, 2596-2621, 2596-2622, 2596-2623, 2596-2624, 2596-2625, 2596-2626, 2596-2627, 2596-2628, 2596-2629, 2596-2630, 2596-2631, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2597-2617, 2597-2618, 2597-2619, 2597-2620, 2597-2621, 2597-2622, 2597-2623, 2597-2624, 2597-2625, 2597-2626, 2597-2627, 2597-2628, 2597-2629, 2597-2630, 2597-2631, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2598-2618, 2598-2619, 2598-2620, 2598-2621, 2598-2622, 2598-2623, 2598-2624, 2598-2625, 2598-2626, 2598-2627, 2598-2628, 2598-2629, 2598-2630, 2598-2631, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2599-2619, 2599-2620, 2599-2621, 2599-2622, 2599-2623, 2599-2624, 2599-2625, 2599-2626, 2599-2627, 2599-2628, 2599-2629, 2599-2630, 2599-2631, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2600-2620, 2600-2621, 2600-2622, 2600-2623, 2600-2624, 2600-2625, 2600-2626, 2600-2627, 2600-2628, 2600-2629, 2600-2630, 2600-2631, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2601-2621, 2601-2622, 2601-2623, 2601-2624, 2601-2625, 2601-2626, 2601-2627, 2601-2628, 2601-2629, 2601-2630, 2601-2631, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2602-2622, 2602-2623, 2602-2624, 2602-2625, 2602-2626, 2602-2627, 2602-2628, 2602-2629, 2602-2630, 2602-2631, 2603-2620, 2603-2621, 2603-2622, 2603-2623, 2603-2624, 2603-2625, 2603-2626, 2603-2627, 2603-2628, 2603-2629, 2603-2630, 2603-2631, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2604-2624, 2604-2625, 2604-2626, 2604-2627, 2604-2628, 2604-2629, 2604-2630, 2604-2631, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2605-2625, 2605-2626, 2605-2627, 2605-2628, 2605-2629, 2605-2630, 2605-2631, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2606-2626, 2606-2627, 2606-2628, 2606-2629, 2606-2630, 2606-2631, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2607-2627, 2607-2628, 2607-2629, 2607-2630, 2607-2631, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2608-2628, 2608-2629, 2608-2630, 2608-2631, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2609-2629, 2609-2630, 2609-2631, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2610-2630, 2610-2631, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2611-2631, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631. In certain aspects, antisense compounds or oligonucleotides target at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases within the aforementioned nucleobase regions.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 50% inhibition: 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 50% inhibition: 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 60% inhibition: 48-63, 150-169, 152-171, 154-169, 154-173, 156-171, 156-175, 158-173, 158-177, 600-619, 1135-1154, 1141-1160, 1147-1166, 1153-1172, 1171-1186, 1173-1188, 1175-1190, 1749-1768, 1763-1782, 1763-1782, 1912-1931, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2197-2212, 2197-2216, 2223-2238, 2225-2240, 2227-2242, 2238-2257, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2550-2569, 2551-2566, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587, 2570-2589, 2571-2588, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2590, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2602, 2586-2604, 2586-2605, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2606, 2588-2607, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2591-2607, 2591-2609, 2591-2610, 2592-2608, 2592-2609, 2592-2611, 2593-2608, 2593-2609, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2630, 2615-2631, 2615-2631, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 60% inhibition: 1685-1704, 1686-1705, 1769-1784, 1871-1890, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1879-1894, 1879-1898, 2808-2827, 3819-3838, 3825-3844, 3831-3850, 3837-3856, 4151-4166, 5890-5909, 5904-5923, 5904-5923, 6406-6425, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6985-7000, 6985-7004, 7122-7141, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7696-7711, 7696-7715, 7786-7801, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7822, 7805-7824, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7825, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7837, 7821-7839, 7821-7840, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7841, 7823-7842, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7844, 7826-7845, 7827-7843, 7827-7844, 7827-7846, 7828-7843, 7828-7844, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, 7846-7862, and 7847-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 70% inhibition: 48-63, 150-169, 152-171, 154-169, 154-173, 156-171, 156-175, 158-173, 158-177, 1135-1154, 1141-1160, 1147-1166, 1171-1186, 1173-1188, 1175-1190, 1749-1768, 1763-1782, 1912-1931, 2193-2212, 2195-2210, 2195-2214, 2197-2212, 2197-2216, 2223-2238, 2225-2240, 2227-2242, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2461-2476, 2461-2480, 2550-2569, 2551-2566, 2552-2571, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2554-2573, 2555-2572, 2555-2574, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587, 2570-2589, 2571-2588, 2571-2590, 2572-2589, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2598, 2580-2599, 2581-2597, 2581-2600, 2582-2598, 2582-2600, 2582-2601, 2583-2599, 2583-2601, 2583-2602, 2584-2600, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2605, 2587-2606, 2588-2604, 2588-2606, 2588-2607, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2609, 2591-2607, 2591-2610, 2592-2611, 2593-2608, 2593-2612, 2594-2609, 2594-2610, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2630, 2615-2631, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 70% inhibition: 1685-1704, 1686-1705, 1769-1784, 1871-1890, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1879-1894, 1879-1898, 3819-3838, 3825-3844, 3831-3850, 4151-4166, 5890-5909, 5904-5923, 5904-5923, 6406-6425, 6983-6998, 6983-7002, 6985-7000, 6985-7004, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7696-7711, 7696-7715, 7786-7801, 7787-7806, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7822, 7805-7824, 7806-7823, 7806-7825, 7807-7824, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7827, 7811-7828, 7811-7830, 7812-7829, 7812-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7833, 7815-7834, 7816-7832, 7816-7835, 7817-7833, 7817-7835, 7817-7836, 7818-7834, 7818-7836, 7818-7837, 7819-7835, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7840, 7822-7841, 7823-7839, 7823-7841, 7823-7842, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7844, 7826-7842, 7826-7845, 7827-7846, 7828-7843, 7828-7847, 7829-7844, 7829-7845, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, 7846-7862, and 7847-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 80% inhibition: 152-171, 154-169, 156-171, 158-173, 1135-1154, 1171-1186, 1173-1188, 1175-1190, 1763-1782, 1912-1931, 2197-2212, 2223-2238, 2225-2240, 2227-2242, 2457-2472, 2459-2474, 2461-2476, 2551-2566, 2553-2570, 2553-2571, 2553-2572, 2554-2573, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2576, 2558-2575, 2558-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2567-2584, 2567-2586, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587, 2571-2588, 2571-2590, 2572-2589, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2592, 2576-2593, 2576-2595, 2577-2594, 2577-2596, 2578-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2600, 2582-2601, 2583-2602, 2584-2603, 2585-2604, 2586-2605, 2587-2606, 2588-2607, 2589-2608, 2590-2606, 2590-2607, 2590-2609, 2591-2610, 2592-2611, 2593-2608, 2593-2612, 2594-2613, 2595-2611, 2595-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2617, 2599-2618, 2600-2615, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2619, 2601-2620, 2602-2618, 2602-2621, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2627, 2609-2624, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 80% inhibition: 1685-1704, 1686-1705, 1873-1892, 1875-1890, 1877-1892, 1879-1894, 3819-3838, 4151-4166, 5904-5923, 6406-6425, 6985-7000, 7692-7707, 7694-7709, 7696-7711, 7786-7801, 7788-7805, 7788-7806, 7788-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809, 7792-7811, 7793-7810, 7793-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7802-7819, 7802-7821, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7822, 7806-7823, 7806-7825, 7807-7824, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7827, 7811-7828, 7812-7829, 7812-7831, 7813-7832, 7814-7833, 7815-7834, 7816-7832, 7816-7835, 7817-7836, 7818-7837, 7819-7838, 7820-7839, 7821-7840, 7822-7841, 7823-7842, 7824-7843, 7825-7841, 7825-7842, 7825-7844, 7826-7845, 7827-7846, 7828-7843, 7828-7847, 7829-7848, 7830-7846, 7830-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7852, 7834-7853, 7835-7850, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7854, 7836-7855, 7837-7853, 7837-7856, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7862, 7844-7859, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7846-7862, and 7847-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 90% inhibition: 154-169, 156-171, 158-173, 1135-1154, 1171-1186, 1173-1188, 1763-1782, 1912-1931, 2223-2238, 2227-2242, 2459-2474, 2461-2476, 2554-2573, 2555-2574, 2560-2577, 2561-2578, 2561-2579, 2562-2581, 2563-2580, 2563-2582, 2564-2581, 2566-2583, 2567-2584, 2568-2585, 2568-2587, 2569-2586, 2570-2587, 2576-2593, 2577-2594, 2577-2596, 2578-2597, 2580-2599, 2581-2600, 2582-2601, 2583-2602, 2584-2603, 2586-2605, 2587-2605, 2587-2606, 2588-2607, 2589-2608, 2590-2607, 2590-2609, 2592-2611, 2595-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2615, 2597-2616, 2598-2613, 2598-2613, 2598-2617, 2599-2614, 2599-2618, 2600-2615, 2600-2619, 2601-2617, 2601-2620, 2602-2621, 2603-2622, 2604-2623, 2605-2621, 2605-2622, 2605-2624, 2606-2625, 2607-2626, 2608-2623, 2608-2625, 2609-2628, 2611-2627, 2611-2630, 2612-2628, 2612-2631, 2613-2629, 2614-2629, 2615-2630, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 90% inhibition: 1685-1704, 1686-1705, 1875-1890, 1877-1892, 1879-1894, 3819-3838, 5904-5923, 6406-6425, 7694-7709, 7696-7711, 7789-7808, 7790-7809, 7795-7812, 7795-7813, 7796-7813, 7796-7814, 7797-7814, 7797-7816, 7798-7815, 7798-7817, 7799-7816, 7801-7818, 7802-7819, 7803-7820, 7803-7822, 7804-7821, 7805-7822, 7811-7828, 7812-7829, 7812-7831, 7813-7832, 7815-7834, 7818-7837, 7819-7838, 7821-7840, 7822-7840, 7822-7841, 7825-7842, 7832-7847, 7832-7848, 7832-7850, 7833-7848, 7833-7852, 7834-7849, 7834-7853, 7835-7850, 7836-7852, 7836-7855, 7837-7856, 7838-7856, 7839-7857, 7839-7858, 7840-7856, 7840-7857, 7840-7859, 7843-7858, 7843-7860, and 7846-7862.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 50% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614, 532632, 532635, 532638, 532639, 532686, 532687, 532688, 532689, 532690, 532691, 532692, 532692, 532693, 532694, 532695, 532696, 532697, 532698, 532699, 532700, 532701, 532702, 532703, 532704, 532705, 532706, 532707, 532770, 532775, 532778, 532780, 532791, 532800, 532809, 532810, 532811, 532917, 532952, 588509, 588510, 588511, 588512, 588513, 588514, 588515, 588516, 588517, 588518, 588519, 588520, 588522, 588523, 588524, 588525, 588527, 588528, 588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588566, 588567, 588568, 588569, 588570, 588571, 588572, 588573, 588574, 588575, 588576, 588577, 588580, 588581, 588585, 588586, 588589, 588590, 588599, 588603, 588606, 588608, 588610, 588614, 588616, 588628, 588631, 588632, 588634, 588636, 588638, 588640, 588645, 588646, 588654, 588656, 588658, 588660, 588662, 588664, 588670, 588672, 588676, 588682, 588688, 588696, 588698, 588807, 588808, 588809, 588813, 588814, 588815, 588819, 588820, 588822, 588823, 588838, 588839, 588840, 588841, 588842, 588846, 588847, 588848, 588849, 588850, 588851, 588852, 588853, 588854, 588855, 588856, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588864, 588865, 588866, 588867, 588868, 588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 588884, 598999, 599000, 599001, 599002, 599003, 599004, 599005, 599006, 599007, 599008, 599009, 599010, 599011, 599012, 599013, 599014, 599015, 599018, 599019, 599023, 599024, 599025, 599026, 599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034, 599035, 599058, 599062, 599063, 599064, 599065, 599070, 599071, 599072, 599073, 599074, 599076, 599077, 599078, 599079, 599080, 599081, 599082, 599083, 599084, 599085, 599086, 599087, 599088, 599089, 599090, 599091, 599092, 599093, 599094, 599095, 599096, 599097, 599098, 599102, 599119, 599123, 599124, 599125, 599126, 599127, 599128, 599132, 599133, 599134, 599135, 599136, 599137, 599138, 599139, 599140, 599141, 599142, 599143, 599144, 599145, 599147, 599148, 599149, 599150, 599151, 599152, 599153, 599154, 599155, 599156, 599157, 599158, 599159, 599178, 599179, 599180, 599181, 599182, 599186, 599187, 599188, 599189, 599190, 599191, 599192, 599193, 599194, 599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599208, 599209, 599210, 599211, 599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599241, 599247, 599248, 599249, 599255, 599256, 599257, 599258, 599260, 599261, 599262, 599263, 599264, 599265, 599266, 599267, 599268, 599269, 599270, 599271, 599272, 599273, 599274, 599275, 599276, 599277, 599278, 599279, 599280, 599297, 599299, 599306, 599307, 599308, 599309, 599311, 599312, 599313, 599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321, 599322, 599323, 599324, 599325, 599326, 599327, 599328, 599329, 599330, 599338, 599349, 599353, 599354, 599355, 599356, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373, 599376, 599378, 599379, 599382, 599383, 599384, 599385, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409, 599410, 599412, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599425, 599426, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443, 599444, 599445, 599446, 599447, 599448, 599450, 599454, 599455, 599456, 599467, 599468, 599469, 599471, 599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599509, 599512, 599515, 599518, 599531, 599541, 599541, 599546, 599547, 599548, 599549, 599550, 599552, 599553, 599554, 599555, 599557, 599558, 599561, 599562, 599563, 599564, 599565, 599566, 599567, 599568, 599569, 599570, 599577, 599578, 599579, 599580, 599581, 599581, 599582, 599584, 599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593, 599594, 599595, 601321, 601322, 601323, 601325, 601327, 601328, 601329, 601330, 601332, 601333, 601334, 601335, 601336, 601337, 601338, 601339, 601341, 601342, 601343, 601344, 601345, 601346, 601347, 601348, 601349, 601362, 601367, 601368, 601369, 601371, 601372, 601373, 601374, 601375, 601377, 601378, 601380, 601381, 601382, 601383, 601384, 601385, 601386, 601387, and 601388.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 50% inhibition of a CFB mRNA, SEQ ID NOs: 12, 30, 33, 36, 37, 84, 85, 86, 87, 88, 89, 90, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 198, 203, 206, 208, 219, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 468, 472, 473, 475, 478, 479, 488, 492, 494, 495, 498, 499, 500, 502, 503, 509, 510, 511, 512, 513, 514, 515, 517, 518, 522, 523, 524, 525, 529, 530, 531, 534, 535, 537, 540, 541, 542, 543, 544, 545, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 563, 564, 565, 569, 570, 572, 573, 577, 588, 589, 590, 591, 592, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 623, 640, 641, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 700, 704, 705, 706, 707, 708, 709, 711, 712, 713, 714, 715, 716, 717, 718, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 758, 759, 760, 761, 762, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 813, 833, 834, 841, 846, 849, 850, 867, and 873.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 60% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614, 532635, 532686, 532687, 532688, 532689, 532770, 532800, 532809, 532810, 532811, 532917, 532952, 588512, 588513, 588514, 588515, 588516, 588517, 588518, 588519, 588522, 588523, 588524, 588525, 588527, 588528, 588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588566, 588567, 588568, 588569, 588570, 588571, 588572, 588573, 588574, 588575, 588576, 588577, 588636, 588638, 588640, 588664, 588676, 588696, 588698, 588807, 588808, 588814, 588815, 588819, 588820, 588840, 588842, 588846, 588847, 588848, 588849, 588850, 588851, 588852, 588853, 588854, 588855, 588856, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588864, 588866, 588867, 588868, 588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 588884, 598999, 599000, 599001, 599002, 599003, 599004, 599005, 599006, 599007, 599008, 599009, 599010, 599011, 599012, 599013, 599014, 599015, 599019, 599024, 599025, 599026, 599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034, 599035, 599064, 599065, 599071, 599072, 599077, 599078, 599079, 599080, 599083, 599084, 599085, 599086, 599087, 599088, 599089, 599090, 599091, 599092, 599093, 599094, 599095, 599096, 599097, 599125, 599126, 599127, 599133, 599134, 599135, 599136, 599138, 599139, 599140, 599141, 599142, 599148, 599149, 599150, 599151, 599152, 599154, 599155, 599156, 599157, 599158, 599159, 599178, 599179, 599180, 599181, 599187, 599188, 599190, 599192, 599193, 599194, 599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599208, 599209, 599210, 599211, 599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599247, 599255, 599256, 599257, 599263, 599264, 599265, 599266, 599270, 599271, 599272, 599273, 599274, 599275, 599276, 599277, 599278, 599279, 599280, 599306, 599307, 599308, 599311, 599312, 599313, 599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321, 599322, 599323, 599324, 599325, 599327, 599328, 599329, 599330, 599349, 599353, 599355, 599356, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373, 599376, 599378, 599379, 599382, 599384, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409, 599410, 599412, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599425, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443, 599444, 599445, 599446, 599447, 599448, 599456, 599467, 599468, 599471, 599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599512, 599531, 599547, 599548, 599549, 599552, 599553, 599554, 599555, 599557, 599558, 599562, 599563, 599564, 599565, 599566, 599567, 599568, 599569, 599570, 599577, 599578, 599579, 599580, 599581, 599582, 599584, 599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593, 599594, 599595, 601323, 601327, 601329, 601332, 601333, 601333, 601334, 601335, 601336, 601338, 601339, 601341, 601342, 601343, 601344, 601345, 601346, 601347, 601348, 601349, 601368, 601369, 601371, 601372, 601374, 601375, 601377, 601378, 601380, 601381, 601382, 601383, 601384, 601385, 601386, 601387, and 601388.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 60% inhibition of a CFB mRNA, SEQ ID NOs: 12, 33, 84, 85, 86, 87, 198, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 472, 473, 513, 514, 515, 531, 537, 541, 542, 543, 544, 545, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 564, 565, 569, 570, 577, 590, 592, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 682, 683, 684, 685, 686, 687, 688, 689, 700, 704, 706, 707, 708, 709, 711, 712, 713, 714, 715, 716, 717, 720, 721, 722, 723, 724, 725, 726, 727, 727, 728, 729, 730, 731, 732, 733, 734, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 758, 759, 760, 761, 767, 768, 770, 772, 773, 774, 775, 775, 776, 776, 777, 777, 778, 779, 780, 781, 782, 783, 783, 784, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 813, 833, 834, 841, 846, 849, and 850.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 70% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614, 532686, 532687, 532688, 532770, 532800, 532809, 532810, 532811, 532917, 532952, 588512, 588513, 588514, 588515, 588516, 588517, 588518, 588524, 588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588568, 588569, 588570, 588571, 588572, 588573, 588574, 588575, 588577, 588636, 588638, 588640, 588696, 588698, 588807, 588814, 588815, 588819, 588842, 588847, 588848, 588849, 588850, 588851, 588852, 588853, 588856, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588866, 588867, 588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 588884, 599000, 599001, 599003, 599004, 599005, 599008, 599009, 599010, 599011, 599014, 599015, 599024, 599025, 599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034, 599072, 599077, 599080, 599085, 599086, 599087, 599088, 599089, 599090, 599091, 599093, 599094, 599095, 599096, 599097, 599125, 599126, 599134, 599138, 599139, 599148, 599149, 599150, 599151, 599152, 599154, 599155, 599156, 599157, 599158, 599187, 599188, 599193, 599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599208, 599210, 599211, 599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599266, 599272, 599272, 599273, 599274, 599275, 599277, 599278, 599279, 599280, 599280, 599306, 599311, 599312, 599313, 599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321, 599322, 599323, 599325, 599327, 599328, 599329, 599330, 599355, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373, 599378, 599379, 599382, 599384, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409, 599410, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443, 599445, 599446, 599447, 599448, 599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599512, 599547, 599548, 599552, 599553, 599554, 599555, 599558, 599562, 599563, 599564, 599566, 599567, 599568, 599569, 599570, 599577, 599578, 599579, 599580, 599581, 599582, 599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593, 599594, 599595, 601332, 601335, 601341, 601343, 601344, 601345, 601346, 601347, 601348, 601349, 601371, 601372, 601380, 601382, 601383, 601384, 601385, 601386, and 601387.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 70% inhibition of a CFB mRNA, SEQ ID NOs: 12, 84, 85, 86, 198, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 402, 403, 404, 405, 407, 408, 410, 411, 412, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 464, 465, 472, 473, 513, 514, 515, 541, 542, 543, 544, 545, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 564, 565, 569, 592, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 645, 646, 647, 648, 649, 650, 653, 654, 655, 656, 659, 660, 662, 663, 664, 665, 666, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 677, 678, 679, 680, 682, 683, 684, 686, 687, 688, 689, 706, 708, 709, 711, 712, 713, 714, 715, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 767, 768, 773, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 793, 794, 795, 797, 798, 799, 813, 833, 834, 841, 846, 849, 867, and 873.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least an 80% inhibition of a CFB mRNA, ISIS NOs: 532686, 532809, 532810, 532811, 532917, 532952, 588512, 588517, 588518, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588571, 588638, 588640, 588696, 588698, 588807, 588814, 588849, 588850, 588851, 588853, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588866, 588867, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 599001, 599024, 599025, 599033, 599086, 599087, 599088, 599089, 599093, 599094, 599095, 599096, 599134, 599139, 599148, 599149, 599151, 599154, 599155, 599156, 599158, 599188, 599195, 599196, 599198, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599212, 599213, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599272, 599273, 599275, 599277, 599278, 599279, 599280, 599311, 599313, 599314, 599316, 599317, 599318, 599320, 599321, 599322, 599323, 599327, 599328, 599329, 599330, 599355, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599371, 599372, 599373, 599378, 599379, 599382, 599384, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599397, 599398, 599399, 599400, 599401, 599403, 599404, 599405, 599407, 599408, 599409, 599410, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599445, 599446, 599447, 599448, 599474, 599476, 599477, 599479, 599481, 599482, 599483, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599547, 599552, 599553, 599554, 599558, 599563, 599567, 599568, 599569, 599570, 599577, 599578, 599581, 599582, 599585, 599587, 599588, 599590, 599591, 599592, 599593, 599594, 601332, 601344, 601345, 601382, 601383, and 601385.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 80% inhibition of a CFB mRNA, SEQ ID NOs: 84, 237, 238, 239, 317, 395, 397, 411, 412, 413, 414, 415, 417, 418, 419, 420, 421, 422, 423, 425, 426, 427, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 472, 473, 514, 515, 542, 543, 544, 545, 546, 547, 550, 551, 552, 553, 554, 555, 556, 557, 564, 595, 599, 600, 601, 602, 603, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 646, 655, 660, 662, 663, 666, 669, 670, 671, 672, 673, 675, 676, 677, 678, 679, 682, 684, 686, 687, 688, 689, 706, 708, 709, 711, 712, 713, 714, 715, 720, 722, 723, 724, 725, 726, 727, 729, 730, 731, 732, 733, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 768, 775, 776, 778, 781, 782, 783, 784, 785, 787, 788, 789, 790, 791, 792, 793, 794, 799, 813, 833, 834, 841, 849, 867, and 873.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 90% inhibition of a CFB mRNA, ISIS NOs: 532686, 532811, 532917, 588536, 588537, 588538, 588539, 588544, 588545, 588546, 588548, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588564, 588638, 588640, 588696, 588698, 588849, 588850, 588851, 588860, 588866, 588867, 588872, 588873, 588874, 588876, 588877, 588878, 588879, 588881, 588883, 599149, 599188, 599203, 599206, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599235, 599236, 599279, 599280, 599314, 599321, 599362, 599378, 599390, 599391, 599398, 599399, 599404, 599413, 599414, 599416, 599419, 599420, 599422, 599435, 599437, 599438, 599441, 599483, 599494, 599508, 599552, 599553, 599554, 599568, 599570, 599577, 599581, 599591, 599592, and 599593.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 90% inhibition of a CFB mRNA, SEQ ID NOs: 84, 238, 239, 317, 412, 413, 420, 421, 426, 434, 436, 437, 438, 439, 440, 442, 443, 444, 445, 446, 448, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 464, 465, 472, 473, 514, 515, 542, 543, 544, 545, 546, 551, 553, 555, 556, 599, 600, 601, 602, 610, 616, 617, 618, 662, 666, 670, 676, 677, 678, 688, 689, 713, 723, 729, 730, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 755, 756, 768, 783, 793, 833, and 867.

In certain embodiments, a compound can comprise or consist of any oligonucleotide targeted to CFB described herein and a conjugate group.

In certain embodiments, a compound comprises a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleotides 2193-2212, 2195-2210, 2457-2476, 2571-2590, 2584-2603, 2588-2607, 2592-2611, 2594-2613, 2597-2616, 2600-2619, or 2596-2611 of SEQ ID NO: 1.

In certain embodiments, a compound comprises a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598.

In certain embodiments, a compound comprises a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide has a nucleobase sequence consisting of any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598.

In certain embodiments, any of the foregoing compounds or oligonucleotides can comprise at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.

In certain aspects, any of the foregoing compounds or oligonucleotides can comprise at least one modified sugar. In certain aspects, at least one modified sugar comprises a 2′-O-methoxyethyl group. In certain aspects, at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH₃)—O-2′ group, a 4′-CH₂-O-2′ group, or a 4′-(CH₂)₂—O-2′group.

In certain aspects, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, or 7 phosphodiester internucleoside linkages.

In certain embodiments, each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, a compound comprises a conjugate group and a modified oligonucleotide comprising:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, or 598.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising or consisting of the sequence recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, or 455, wherein the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of five linked nucleosides; and

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a single-stranded modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, or 455, wherein the oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of five linked nucleosides; and

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of ISIS 588540 and a conjugate group. In certain embodiments, ISIS 588540 has the following chemical structure:

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising or consisting of the sequence recited in SEQ ID NO: 549, wherein the modified oligonucleotide comprises

a gap segment consisting often linked deoxynucleosides;

a 5′ wing segment consisting of three linked nucleosides; and

a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain aspects, the modified oligonucleotide has a nucleobase sequence comprising or consisting of the sequence recited in SEQ ID NO: 598, wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of three linked nucleosides; and

a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment comprises a 2′-O-methoxyethyl sugar, 2′-O-methoxyethyl sugar, and cEt sugar in the 5′ to 3′ direction; wherein the 3′ wing segment comprises a cEt sugar, cEt sugar, and 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing embodiments, the compound or oligonucleotide can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding CFB.

In any of the foregoing embodiments, the compound or oligonucleotide can be single-stranded.

In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3′ end of the modified oligonucleotide. In certain embodiments, the conjugate group comprises at least one N-Acetylgalactosamine (GalNAc), at least two N-Acetylgalactosamines (GalNAcs), or at least three N-Acetylgalactosamines (GalNAcs).

In certain embodiments, a compound having the following chemical structure comprises or consists of ISIS 588540 with a 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

In certain embodiments, a compound comprises or consists of SEQ ID NO: 440, 5′-GalNAc, and chemical modifications as represented by the following chemical structure:

wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R² together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and R¹ and R² are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—; And for each pair of R³ and R⁴ on the same ring, independently for each ring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³ and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—; And R⁵ is selected from H and —CH₃; And Z is selected from S— and O—.

In certain embodiments, a compound comprises ISIS 696844. In certain embodiments, a compound consists of ISIS 696844. In certain embodiments, ISIS 696844 has the following chemical structure:

In certain embodiments, a compound comprises ISIS 696845. In certain embodiments, a compound consists of ISIS 696845. In certain embodiments, ISIS 696845 has the following chemical structure:

In certain embodiments, a compound comprises ISIS 698969. In certain embodiments, a compound consists of ISIS 698969. In certain embodiments, ISIS 698969 has the following chemical structure:

In certain embodiments, a compound comprises ISIS 698970. In certain embodiments, a compound consists of ISIS 698970. In certain embodiments, ISIS 698970 has the following chemical structure:

Certain embodiments provide compositions comprising any of the compounds comprising or consisting of a modified oligonucleotide targeted to CFB or salt thereof and a conjugate group, and at least one of a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC₅₀ of less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 45 nM, less than 40 nM, less than 35 nM, less than 30 nM, less than 25 nM, or less than 20 nM.

In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase an ALT or AST value of no more than 4 fold, 3 fold, or 2 fold over saline treated animals or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2%. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over saline treated animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over saline treated animals.

Certain embodiments provide a composition comprising the compound of any of the aforementioned embodiments or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain aspects, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain aspects, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain aspects, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature or about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.

In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound or composition described herein, thereby treating, preventing, or ameliorating the disease. In certain aspects, the complement alternative pathway is activated greater than normal. In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970.

In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD) in a subject comprises administering to the subject a compound or composition described herein, thereby treating, preventing, or ameliorating AMD. In certain aspects, the complement alternative pathway is activated greater than normal. In certain aspects, the AMD is wet AMD. In certain aspects, the AMD is dry AMD, such as Geographic Atrophy. In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration in a subject, such as age-related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy comprises administering to the subject a a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy in a subject comprises administering to the subject a comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy in a subject comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the compound or composition is administered to the subject parenterally.

In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound or composition described herein, thereby treating, preventing, or ameliorating the kidney disease. In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the complement alternative pathway is activated greater than normal. In certain aspects, the kidney disease is lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or a typical hemolytic uremic syndrome (aHUS), or any combination thereof. In certain aspects, the kidney disease is associated with C3 deposits, such as C3 deposits in the glomerulus. In certain aspects, the kidney disease is associated with lower than normal circulating C3 levels, such as serum or plasma C3 levels. In certain aspects, administering the compound or composition reduces or inhibits accumulation of ocular C3 levels, such as C3 protein levels. In certain aspects, administering the compound or composition reduces the level of ocular C3 deposits or inhibits accumulation of ocular C3 deposits. In certain aspects, the compound or composition is administered to the subject parenterally. In certain aspects, administering the compound or composition reduces or inhibits accumulation of C3 levels in the kidney, such as C3 protein levels. In certain aspects, administering the compound or composition reduces the level of kidney C3 deposits or inhibits accumulation of kidney C3 deposits, such as C3 levels in the glomerulus. In certain aspects, the subject is identified as having or at risk of having a disease associated with dysregulation of the complement alternative pathway, for example by detecting complement levels or membrane-attack complex levels in the subject's blood and/or performing a genetic test for gene mutations of complement factors associated with the disease.

In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a compound or composition described herein to the subject, thereby inhibiting expression of CFB in the subject. In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, administering the compound or composition inhibits expression of CFB in the eye. In certain aspects, the subject has, or is at risk of having, age related macular degeneration (AMD), such as wet AMD and dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. Geographic Atrophy is considered an advanced form of dry AMD involving degeneration of the retina. In certain aspects, administering the compound or composition inhibits expression of CFB in the kidney, such as in the glomerulus. In certain aspects, the subject has, or is at risk of having, lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or a typical hemolytic uremic syndrome (aHUS), or any combination thereof.

In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a compound or composition described herein to the subject, thereby reducing or inhibiting accumulation of C3 deposits in the eye of the subject. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the subject has, or is at risk of having, age related macular degeneration (AMD), such as wet AMD and dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. In certain aspects, the compound or composition is administered to the subject parenterally.

In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a compound or composition described herein to the subject, thereby reducing or inhibiting accumulation of C3 deposits in the kidney of the subject. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the subject has, or is at risk of having, lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or a typical hemolytic uremic syndrome (aHUS), or any combination thereof. In certain aspects, the compound or composition is administered to the subject parenterally.

Certain embodiments are drawn to use of a compound or composition described herein for treating a disease associated with dysregulation of the complement alternative pathway. Certain embodiments are drawn to use of a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808, for treating a disease associated with dysregulation of the complement alternative pathway. Certain embodiments are drawn to use of a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598, for treating a disease associated with dysregulation of the complement alternative pathway. Certain embodiments are drawn to use of a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970 for treating a disease associated with dysregulation of the complement alternative pathway. In certain aspects, the complement alternative pathway is activated greater than normal. In certain aspects, the disease is macular degeneration, such as age related macular degeneration (AMD), which can be wet AMD or dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. In certain aspects, the disease is a kidney disease such as lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or a typical hemolytic uremic syndrome (aHUS), or any combination thereof. In certain aspects, the compound or composition is administered to the subject parenterally.

In certain embodiments, a compound or composition described herein is administered parenterally. For example, in certain embodiments the compound or composition can be administered through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to an CFB nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated.

In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.

Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound targeted to a CFB nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.

i. ssRNA Compounds

In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5′-terminal end. In certain such embodiments, the 5′-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5′-phosphate). In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. Certain modified 5′-terminal nucleosides may be found in the art, for example in WO/2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound has Formula IIc:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula IIc to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄ R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C1-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certain embodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certain embodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certain embodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ are each, independently, H or halogen. In certain embodiments, Q₁ and Q₂ is H and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protected thiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R_(c) are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁), O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) or O(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ are each, independently, H or C₁-C₆ alkyl. In certain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certain embodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G is O(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2′-F and 2′—OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB)_(x)A_(y) wherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA; ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5′ terminal nucleoside, such as those of formula IIc or IIe.

In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif: -(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-

wherein: A is a first type of modifed nucleosde;

B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(AB)_(x)A_(y)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modifed nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it.

Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(A)_(x)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modifed nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating PO/PS 8 PS

ii. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.

The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is CFB. In certain embodiment, the degradation of the targeted CFB is facilitated by an activated RISC complex that is formed with compositions of the invention.

Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC.

The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of several embodiments provided herein can target CFB by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., “hairpin” or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.

In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5′ phosphoryl group of a nucleotide becomes linked to the 2′ hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2′ position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode Complement Factor B (CFB) include, without limitation, the following: GENBANK Accession No. NM_001710.5 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No NW_001116486.1 truncated from nucleotides 536000 to 545000 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. XM_001113553.2 (incorporated herein as SEQ ID NO: 4), or GENBANK Accession No. NM_008198.2 (incorporated herein as SEQ ID NO: 5).

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a CFB nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a CFB nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a CFB nucleic acid).

Non-complementary nucleobases between an antisense compound and a CFB nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a CFB nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a CFB nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a CFB nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a CFB nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a CFB nucleic acid, or specified portion thereof.

The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′,3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a CFB nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

In certain embodiments, oligonucleotides comprise one or more methylphosponate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosponate linkages. In certain embodiments, one methylphosponate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂ are each independently H, C₁-C₁₂ alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R), where each R₁, R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as constrained ethyl or cEt) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see published International Application WO 2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Zhou et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; 8,530,640; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 61/026,995 and 61/097,787; Published PCT International applications; WO 2009/067647; WO 2011/017521; WO 2010/036698 WO 1999/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)_(x)—, —C(═NR_(a))—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′,4′-(CH₂)₂-2′,4′—(CH₂)₃-2′,4′-CH₂—O-2′,4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′— wherein each R is, independently, H, a protecting group or C1-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the 3-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy (4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F) methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA and (K) vinyl BNA as depicted below:

wherein Bx is the base moiety and R is independently H, a protecting group, C₁-C₁₂ alkyl or C₁-C₁₂ alkoxy.

In certain embodiments, bicyclic nucleosides are provided having Formula I:

wherein:

Bx is a heterocyclic base moiety; -Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(Re)—CH₂—, —C(═O)—N(Re)—CH₂—, —CH₂—O—N(Re)—, —CH₂—N(Re)—O— or —N(Re)—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃, OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl or substituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q₁ is, independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J, O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q₁ and q_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O [(CH₂)_(n)O]_(m)CH₃, O(CH₂)NH₂, O(CH₂)CH₃, O(CH₂)F, O(CH₂)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)CH₃]2, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:

In certain embodiments, sugar surrogates are selected having Formula VII:

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T_(a) and T_(b) is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T_(a) and T_(b) is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁ and R₂ is fluoro. In certain embodiments, R₁ is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modifed morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horváth et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X: Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugar substituent group.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH₃ group at the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a CFB nucleic acid comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to a CFB nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Conjugated Antisense Compounds

In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously. See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005). Such receptors are expressed on liver cells, particularly hepatocytes. Further, it has been shown that compounds comprising clusters of three N-acetylgalactosamine (GalNAc) ligands are capable of binding to the ASGP-R, resulting in uptake of the compound into the cell. See e.g., Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). Accordingly, conjugates comprising such GalNAc clusters have been used to facilitate uptake of certain compounds into liver cells, specifically hepatocytes. For example it has been shown that certain GalNAc-containing conjugates increase activity of duplex siRNA compounds in liver cells in vivo. In such instances, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex. Since the sense strand is discarded before the antisense strand ultimately hybridizes with the target nucleic acid, there is little concern that the conjugate will interfere with activity. Typically, the conjugate is attached to the 3′ end of the sense strand of the siRNA. See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups described herein are more active and/or easier to synthesize than conjugate groups previously described.

In certain embodiments of the present invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to RNase H based antisense compounds and antisense compounds that alter splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain attached to the antisense compound long enough to provide benefit (improved uptake into cells) but then should either be cleaved, or otherwise not interfere with the subsequent steps necessary for activity, such as hybridization to a target nucleic acid and interaction with RNase H or enzymes associated with splicing or splice modulation. This balance of properties is more important in the setting of single-stranded antisense compounds than in siRNA compounds, where the conjugate may simply be attached to the sense strand. Disclosed herein are conjugated single-stranded antisense compounds having improved potency in liver cells in vivo compared with the same antisense compound lacking the conjugate. Given the required balance of properties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavable moiety. As noted, without wishing to be bound by mechanism, it is logical that the conjugate should remain on the compound long enough to provide enhancement in uptake, but after that, it is desirable for some portion or, ideally, all of the conjugate to be cleaved, releasing the parent compound (e.g., antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments take advantage of endogenous nucleases in the cell by attaching the rest of the conjugate (the cluster) to the antisense oligonucleotide through a nucleoside via one or more cleavable bonds, such as those of a phosphodiester linkage. In certain embodiments, the cluster is bound to the cleavable nucleoside through a phosphodiester linkage. In certain embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three cleavable nucleosides. In such embodiments, such cleavable nucleosides are linked to one another, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage). Certain conjugates herein do not comprise a cleavable nucleoside and instead comprise a cleavable bond. It is shown that that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is vulnerable to cleavage in the cell (a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are ultimately metabolized to a more active form. For example, conjugated antisense compounds are cleaved to remove all or part of the conjugate resulting in the active (or more active) form of the antisense compound lacking all or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of an oligonucleotide. Certain such 5′-conjugates are cleaved more efficiently than counterparts having a similar conjugate group attached at the 3′ end. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, oligonucleotides comprising a conjugate at the 5′ end have greater efficacy than oligonucleotides comprising a conjugate at the 3′ end (see, for example, Examples 56, 81, 83, and 84). Further, 5′-attachment allows simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3′ to 5′ direction. To make a 3′-conjugated oligonucleotide, typically one attaches a pre-conjugated 3′ nucleoside to the solid support and then builds the oligonucleotide as usual. However, attaching that conjugated nucleoside to the solid support adds complication to the synthesis. Further, using that approach, the conjugate is then present throughout the synthesis of the oligonucleotide and can become degraded during subsequent steps or may limit the sorts of reactions and reagents that can be used. Using the structures and techniques described herein for 5′-conjugated oligonucleotides, one can synthesize the oligonucleotide using standard automated techniques and introduce the conjugate with the final (5′-most) nucleoside or after the oligonucleotide has been cleaved from the solid support.

In view of the art and the present disclosure, one of ordinary skill can easily make any of the conjugates and conjugated oligonucleotides herein. Moreover, synthesis of certain such conjugates and conjugated oligonucleotides disclosed herein is easier and/or requires few steps, and is therefore less expensive than that of conjugates previously disclosed, providing advantages in manufacturing. For example, the synthesis of certain conjugate groups consists of fewer synthetic steps, resulting in increased yield, relative to conjugate groups previously described. Conjugate groups such as GalNAc3-10 in Example 46 and GalNAc3-7 in Example 48 are much simpler than previously described conjugates such as those described in U.S. Pat. No. 8,106,022 or U.S. Pat. No. 7,262,177 that require assembly of more chemical intermediates. Accordingly, these and other conjugates described herein have advantages over previously described compounds for use with any oligonucleotide, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or two GalNAc ligands. As shown, such conjugates groups improve activity of antisense compounds. Such compounds are much easier to prepare than conjugates comprising three GalNAc ligands. Conjugate groups comprising one or two GalNAc ligands may be attached to any antisense compounds, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially alter certain measures of tolerability. For example, it is shown herein that conjugated antisense compounds are not more immunogenic than unconjugated parent compounds. Since potency is improved, embodiments in which tolerability remains the same (or indeed even if tolerability worsens only slightly compared to the gains in potency) have improved properties for therapy.

In certain embodiments, conjugation allows one to alter antisense compounds in ways that have less attractive consequences in the absence of conjugation. For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with phosphodiester linkages results in improvement in some measures of tolerability. For example, in certain instances, such antisense compounds having one or more phosphodiester are less immunogenic than the same compound in which each linkage is a phosphorothioate. However, in certain instances, as shown in Example 26, that same replacement of one or more phosphorothioate linkages with phosphodiester linkages also results in reduced cellular uptake and/or loss in potency. In certain embodiments, conjugated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the conjugated full-phosphorothioate counterpart. In fact, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides comprising a conjugate and at least one phosphodiester internucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since conjugation results in substantial increases in uptake/potency a small loss in that substantial gain may be acceptable to achieve improved tolerability. Accordingly, in certain embodiments, conjugated antisense compounds comprise at least one phosphodiester linkage.

In certain embodiments, conjugation of antisense compounds herein results in increased delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver tissue. However, in certain embodiments, that increased delivery alone does not explain the entire increase in activity. In certain such embodiments, more compound enters hepatocytes. In certain embodiments, even that increased hepatocyte uptake does not explain the entire increase in activity. In such embodiments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in hepatocytes versus non-parenchymal cells. This enrichment is beneficial for oligonucleotides that target genes that are expressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result in reduced kidney exposure. For example, as shown in Example 20, the concentrations of antisense oligonucleotides comprising certain embodiments of GalNAc-containing conjugates are lower in the kidney than that of antisense oligonucleotides lacking a GalNAc-containing conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the formula: A-B—C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group “D” branches as many times as is necessary to accommodate the number of (E-F) groups as indicated by “q”. Thus, where q=1, the formula is: A-B—C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

The present disclosure provides the following non-limiting numbered embodiments:

Embodiment 1

The conjugated antisense compound of any of embodiments 1179 to 1182, wherein the tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

Embodiment 2

The conjugated antisense compound of any of embodiments 1179 to 1182, wherein the tether has the structure:

Embodiment 3

The conjugated antisense compound of any of embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has a structure selected from among:

Embodiment 4

The conjugated antisense compound of any of embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

Embodiment 5

The conjugated antisense compound of any of embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has the structure:

In embodiments having more than one of a particular variable (e.g., more than one “m” or “n”), unless otherwise indicated, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another.

i. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety attaches to the antisense oligonucleotide. In certain such embodiments, the cleavable moiety attaches directly to the cell-targeting moiety. In certain such embodiments, the cleavable moiety attaches to the conjugate linker. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is 2′-deoxy nucleoside that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2′-deoxy adenosine that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2′-deoxy adenosine that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to a 2′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached to the linker by either a phosphodiester or a phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after the complex has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved thereby releasing the active antisense oligonucleotide. While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more nucleases within the cell. In certain embodiments, the one or more nucleases cleave the phosphodiester linkage between the cleavable moiety and the linker. In certain embodiments, the cleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following:

ii. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. In certain such embodiments, the linker is covalently bound to the cleavable moiety. In certain such embodiments, the linker is covalently bound to the antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell-targeting moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support. In certain embodiments, the linker further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support and further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker further comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the linear group comprises groups selected from alkyl and ether groups. In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group includes at least one neutral linking group. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the cleavable moiety. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the antisense oligonucleotide. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety and a solid support. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety, a solid support and a protein binding moiety. In certain embodiments, the linear group includes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalently attached to a scaffold group. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide and ether groups. In certain embodiments, the scaffold includes at least one mono or polycyclic ring system. In certain embodiments, the scaffold includes at least two mono or polycyclic ring systems. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety and the linker. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a solid support. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a protein binding moiety. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker, a protein binding moiety and a solid support. In certain embodiments, the scaffold group includes one or more cleavable bond.

In certain embodiments, the linker includes a protein binding moiety. In certain embodiments, the protein binding moiety is a lipid such as for example including but not limited to cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

-   -   wherein each L is, independently, a phosphorus linking group or         a neutral linking group; and each n is, independently, from 1 to         20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

iii. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targeting moieties. Certain such cell-targeting moieties increase cellular uptake of antisense compounds. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, and one or more ligand. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, one or more ligand and one or more cleavable bond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group attaches the conjugate linker. In certain embodiments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group attaches the antisense oligonucleotide. In certain embodiments, the branching group is covalently attached to the linker and each of the tethered ligands. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the branching group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system. In certain embodiments, the branching group comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a branching group.

In certain embodiments, a branching group has a structure selected from among:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

wherein A₁ is O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group through either an amide or an ether group. In certain embodiments, the tether is attached to the branching group through a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises from about 10 to about 18 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises about 13 atoms in chain length.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

wherein L is either a phosphorus linking group or a neutral linking group;

Z₁ is C(═O)O—R₂;

Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;

R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and

each m₁ is, independently, from 0 to 20 wherein at least one m₁ is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

wherein Z₂ is H or CH₃; and

-   -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group. In certain embodiments, a tether does not comprise any amide bonds. In certain embodiments, a tether comprises a phosphorus linking group and does not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands wherein each ligand is covalently attached to a tether. In certain embodiments, each ligand is selected to have an affinity for at least one type of receptor on a target cell. In certain embodiments, ligands are selected that have an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In certain embodiments, the targeting moiety comprises 3 N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, modified carbohydrate, multivalent carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, α-D-galactosamine, N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-1-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-β-D-glucopyranose, Methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. In certain embodiments, “N-acetyl galactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the β-form: 2-(Acetylamino)-2-deoxy-3β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these structures are intended to include the other form as well. For example, where the structure for an α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments, In certain preferred embodiments, the β-form 2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected from among:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected from among:

In certain embodiments one or more ligand has a structure selected from among:

In certain embodiments one or more ligand has a structure selected from among:

i. Certain Conjugates

In certain embodiments, conjugate groups comprise the structural features above. In certain such embodiments, conjugate groups have the following structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of six to eleven consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether often consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to eleven consecutively bonded atoms and wherein the tether comprises exactly one amide bond.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted or unsubstituted alkyl group, or a group comprising exactly one ether or exactly two ethers, an amide, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of eight consecutively bonded atoms, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein the tether comprises exactly one amide bond, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms and wherein the tether consists of an amide bond and a substituted or unsubstituted C₂-C₁₁ alkyl group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl group, or a group comprising an ether, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein n is 4, 5, 6, 7, or 8.

In certain embodiments, conjugates do not comprise a pyrrolidine.

a Certain Conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′,3′, of 5′ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure: A-B—C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A-C-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain such embodiments, the branching group comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′,3′, of 5′ position of the nucleoside.

In certain embodiments, a conjugated antisense compound has the following structure: A-B—C

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′,3′, of 5′ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure: A-C

E-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A-B-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A-D

E-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

Representative United States patents, United States patent application publications, and international patent application publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, U.S. Pat. No. 5,994,517, U.S. Pat. No. 6,300,319, U.S. Pat. No. 6,660,720, U.S. Pat. No. 6,906,182, U.S. Pat. No. 7,262,177, U.S. Pat. No. 7,491,805, U.S. Pat. No. 8,106,022, U.S. Pat. No. 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, each of which is incorporated by reference herein in its entirety.

Representative publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, BIESSEN et al., “The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., “Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE et al., “New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes” Bioorganic & Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNase H based oligonucleotide (such as a gapmer) or a splice modulating oligonucleotide (such as a fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain embodiments a conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998/013381; WO2011/038356; WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.

Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Certain Indications

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject by administration of a CFB specific inhibitor, such as an antisense compound targeted to CFB.

Examples of renal diseases associated with dysregulation of the complement alternative pathway treatable, preventable, and/or ameliorable with the methods provided herein include C3 glomerulopathy, a typical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD; also known as MPGN Type II or C3Neph), and CFHR5 nephropathy.

Additional renal diseases associated with dysregulation of the complement alternative pathway treatable, preventable, and/or ameliorable with the methods provided herein include IgA nephropathy; mesangiocapillary (membranoproliferative) glomerulonephritis (MPGN); autoimmune disorders including lupus nephritis and systemic lupus erythematosus (SLE); infection-induced glomerulonephritis (also known as Postinfectious glomerulonephritis); and renal ischemia-reperfusion injury, for example post-transplant renal ischemia-reperfusion injury.

Examples of non-renal disorders associated with dysregulation of the complement alternative pathway treatable and/or preventable with the methods provided herein include ocular diseases such as macular degeneration, for example age-related macular degeneration (AMD), including wet AMD and dry AMD, such as Geographic Atrophy; neuromyelitis optica; corneal disease, such as corneal inflammation; autoimmune uveitis; and diabetic retinopathy. It has been reported that complement system is involved in ocular diseases. Jha P, et al., Mol Immunol (2007) 44(16): 3901-3908. Additional examples of non-renal disorders associated with dysregulation of the complement alternative pathway treatable and/or preventable with the methods provided herein include ANCA-assocaited vasculitis, antiphospholipid syndrome (also known as antiphospholipid antibody syndrome (APS)), asthma, rheumatoid arthritis, Myasthenia Gravis, and multiple sclerosis.

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a renal disease associated with dysregulation of the complement alternative pathway in a subject by administration of a CFB specific inhibitor, such as an antisense compound targeted to CFB. In certain aspects, the renal disease is lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or a typical hemolytic uremic syndrome (aHUS), or any combination thereof.

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD), in a subject by administration of a CFB specific inhibitor, such as an antisense compound targeted to CFB. In certain aspects, the AMD is wet AMD or dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. Studies have demonstrated the association of complement alternative pathway dysregulation and AMD. Complement components are common constituents of ocular drusen, the extracellular material that accumulates in the macula of AMD patients. Furthermore, it has been reported that CFH and CFB variants account for nearly 75% of AMD cases in northern Europe and North America. It has also been found that a specific CFB polymorphism confers protection against AMD. Patel, N. et al., Eye (2008) 22(6):768-76. Additionally, CFB homozygous null mice have lower complement pathway activity, exhibit smaller ocular lesions, and choroidal neovascularization (CNV) after laser photocoagulation. Rohrer, B. et al., Invest Ophthalmol Vis Sci. (2009) 50(7):3056-64. Furthermore, CFB siRNA treatment protects mice from laser induced CNV. Bora, N S et al., J Immunol. (2006) 177(3):1872-8. Studies have also shown that the kidney and eye share developmental pathways and structural features including basement membrane collagen IV protomer composition and vascularity. Savige et al., J Am Soc Nephrol. (2011) 22(8):1403-15. There is evidence that the complement pathway is involved in renal and ocular diseases. For instance, inherited complement regulatory protein deficiency causes predisposition to a typical hemolytic uremic syndrome and AMD. Richards A et al., Adv Immunol. (2007) 96:141-77. Additionally, chronic kidney disease has been associated with AMD. Nitsch, D. et al., Ophthalmic Epidemiol. (2009) 16(3):181-6; Choi, J. et al, Ophthalmic Epidemiol. (2011) 18(6):259-63. Dense deposit disease (DDD), a kidney disease associated with dysregulated complement alternative pathway, is characterized by acute nephritic syndrome and ocular drusen. Cruz and Smith, GeneReviews (2007) July 20. Moreover, mice harboring genetic deletion of a component of the complement alternative pathway have coexisting renal and ocular disease phenotypes. It has been reported that CFH homozygous null mice develop DDD and present retinal abnormalities and visual dysfunction. Pickering et al., Nat Genet. (2002) 31(4):424-8. Mouse models of renal diseases associated with dysregulation of the complement alternative pathway are also accepted as models of AMD. Pennesi M E et al., Mol Aspects Med (2012) 33:487-509. CFH null mice, for example, are an accepted model for renal diseases, such as DDD, and AMD. Furthermore, it has been reported that AMD is associated with the systemic source of complement factors, which accumulate locally in the eye to drive alternative pathway complement activation. Loyet et al., Invest Ophthalmol Vis Sci. (2012) 53(10):6628-37.

EXAMPLES

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites, Compounds 1, 1a and 2

Bx is a heterocyclic base;

Compounds 1, 1a and 2 were prepared as per the procedures well known in the art as described in the specification herein (see Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT International Applications (WO 2011/115818, WO 2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO 2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

Compounds 3 (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem., 1991, 34, 2692).

Example 3: Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 5: Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5.

Example 7: Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc3-1 at the 3′ Terminus, Compound 29

Wherein the protected GalNAc₃-1 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-1 (GalNAc₃-1_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-1_(a) has the formula:

The solid support bound protected GalNAc₃-1, Compound 25, was prepared as per the procedures illustrated in Example 7. Oligomeric Compound 29 comprising GalNAc₃-1 at the 3′ terminus was prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc3-1 at the 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34 comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 11: Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustrated in Examples 2, 4, and 5. Compound 35 is prepared using similar procedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

Example 12: Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated in Examples 5 and 11. Compound 41 is prepared using similar procedures published in WO 2009082607.

Example 14: Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

Compound 46 is commercially available.

Example 16: Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General Method for the Preparation of Conjugated ASOs Comprising GalNAc₃-1 at the 3′ Position Via Solid Phase Techniques (Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and ^(m)c residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on an GalNAc₃-1 loaded VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered 4 fold excess over the loading on the solid support and phosphoramidite condensation was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing dimethoxytrityl (DMT) group from 5′-hydroxyl group of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1:1 (v/v) mixture of triethylamine and acetonitrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesized using standard oligonucleotide synthesis procedures well known in the art.

Using these methods, three separate antisense compounds targeting ApoC III were prepared. As summarized in Table 17, below, each of the three antisense compounds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages; ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1 conjugated at its 3′end; and ISIS 647536 is the same as ISIS 647535 except that certain internucleoside linkages of that compound are phosphodiester linkages. As further summarized in Table 17, two separate antisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a 2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc₃-1 at its 3′-end.

TABLE 17 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed ID ASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds) T_(es)T_(es)T_(es)A_(es)T_(e) ApoC 7165.4 7164.4 821 304801 III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9239.5 9237.8 822 647535 GalNAc ₃ -1 _(a) III ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9142.9 9140.8 822 647536 GalNAc ₃ -1 _(a) III ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) SRB-1 4647.0 4646.4 823 440762 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1 _(a) SRB-1 6721.1 6719.4 824 651900

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having the structure shown previously in Example 9. Note that GalNAc₃-1 comprises a cleavable adenosine which links the ASO to remainder of the conjugate, which is designated “GalNAc₃-1_(a).” This nomenclature is used in the above table to show the full nucleobase sequence, including the adenosine, which is part of the conjugate. Thus, in the above table, the sequences could also be listed as ending with “GalNAc₃-1” with the “A_(do)” omitted. This convention of using the subscript “a” to indicate the portion of a conjugate group lacking a cleavable nucleoside or cleavable moiety is used throughout these Examples. This portion of a conjugate group lacking the cleavable moiety is referred to herein as a “cluster” or “conjugate cluster” or “GalNAc₃ cluster.” In certain instances it is convenient to describe a conjugate group by separately providing its cluster and its cleavable moiety.

Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III in huApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25, 0.75, 2.25 or 6.75 μmol/kg or with PBS as a control. Each treatment group consisted of 4 animals. Forty-eight hours after the administration of the last dose, blood was drawn from each mouse and the mice were sacrificed and tissues were collected.

ApoC III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of ApoC III mRNA levels for each treatment group, normalized to PBS-treated control and are denoted as “% PBS”. The half maximal effective dosage (ED₅₀) of each ASO is also presented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relative to the PBS control. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Dose ED₅₀ SEQ (μmol/ % (μmol/ Internucleoside ID ASO kg) PBS kg) 3′ Conjugate linkage/Length No. PBS 0 100 — — — ISIS 0.08 95 0.77 None PS/20 821 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074 GalNAc ₃ -1 PS/20 822 647535 0.75 15 2.25 17 6.75 8 ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using procedures reported by Graham et al, Circulation Research, published online before print Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed without dilution using an Olympus Clinical Analyzer and a commercially available turbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical, Seattle, Wash.). The assay protocol was performed as described by the vendor.

As shown in the Table 19 below, both antisense compounds reduced ApoC III protein relative to the PBS control. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Dose ED₅₀ SEQ (μmol/ % (μmol/ 3′ Internucleoside ID ASO kg) PBS kg) Conjugate Linkage/Length No. PBS 0 100 — — — ISIS 0.08 86 0.73 None PS/20 821 304801 0.75 51 2.25 23 6.75 13 ISIS 0.08 72 0.19 GalNAc ₃ -1 PS/20 822 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol. 37: 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical analyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice and are denoted as “% PBS”. Results are presented in Table 20. As illustrated, both antisense compounds lowered triglyceride levels. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 20 Effect of ASO treatment on triglyceride levels in transgenic mice Dose ED₅₀ SEQ (μmol/ % (μmol/ 3′ Internucleoside ID ASO kg) PBS kg) Conjugate Linkage/Length No. PBS 0 100 — — — ISIS 0.08 87 0.63 None PS/20 821 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc ₃ -1 PS/20 822 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and of different fractions of cholesterol (HDL and LDL). Results are presented in Tables 21 and 22. As illustrated, both antisense compounds lowered total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoC III.

TABLE 21 Effect of ASO treatment on total cholesterol levels in transgenic mice Total SEQ Dose Cholesterol 3′ Internucleoside ID ASO (μmol/kg) (mg/dL) Conjugate Linkage/Length No. PBS 0 257 — — ISIS 0.08 226 None PS/20 821 304801 0.75 164 2.25 110 6.75 82 ISIS 0.08 230 GalNAc ₃ -1 PS/20 822 647535 0.75 82 2.25 86 6.75 99

TABLE 22 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL SEQ (μmol/ (mg/ LDL 3′ Internucleoside ID ASO kg) dL) (mg/dL) Conjugate Linkage/Length No. PBS 0 17 28 — — ISIS 0.08 17 23 None PS/20 821 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS 0.08 21 21 GalNAc ₃ -1 PS/20 822 647535 0.75 44 2 2.25 50 2 6.75 58 2 Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were analyzed on MSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS 304801 and 647535 was measured and the results are provided in Table 23. As illustrated, liver concentrations of total full-length antisense compounds were similar for the two antisense compounds. Thus, even though the GalNAc₃-1-conjugated antisense compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated EC₅₀ (provided in Table 23) confirms that the observed increase in potency of the conjugated compound cannot be entirely attributed to increased accumulation. This result suggests that the conjugate improved potency by a mechanism other than liver accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.

The results also show that the concentration of GalNAc₃-1 conjugated antisense compound in the kidney is lower than that of antisense compound lacking the GalNAc conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly, for non-kidney targets, kidney accumulation is undesired. These data suggest that GalNAc₃-1 conjugation reduces kidney accumulation.

TABLE 23 PK analysis of ASO treatment in transgenic mice Dose SEQ (μmol/ Liver Kidney Liver EC₅₀ 3′ Internucleoside ID ASO kg) (μg/g) (μg/g) (μg/g) Conjugate Linkage/Length No. ISIS 0.1 5.2 2.1 53 None PS/20 821 304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.8 0.7 3.8 GalNAc ₃ -1 PS/20 822 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8 237.2 179.3

Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass spectrometry analysis. The cleavage sites and structures of the observed metabolites are shown below. The relative % of full length ASO was calculated using standard procedures and the results are presented in Table 23a. The major metabolite of ISIS 647535 was full-length ASO lacking the entire conjugate (i.e. ISIS 304801), which results from cleavage at cleavage site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other cleabable bonds such as esters, peptides, disulfides, phosphoramidates or acyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can be cleaved by enzymes inside the cell, or which may cleave in the reductive environment of the cytosol, or which are labile to the acidic pH inside endosomes and lyzosomes, can also be useful.

TABLE 23a Observed full length metabolites of ISIS 647535 Cleavage Relative Metabolite ASO site % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3 GalNAc + 1 5-hydroxy-pentanoic 5 acid tether] ISIS 647535 minus D 9.9 [2 GalNAc + 2 5-hydroxy-pentanoic 6 acid tether] ISIS 647535 minus D 9.8 [3 GalNAc + 3 5-hydroxy-pentanoic acid tether]

Example 21: Antisense Inhibition of Human ApoC III in Human ApoC III Transgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III and described in Table 17, were further evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once at the dosage shown below with ISIS 304801, 647535 or 647536 (described above) or with PBS treated control. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA and protein levels in the liver; plasma triglycerides; and cholesterol, including HDL and LDL fractions were assessed as described above (Example 20). Data from those analyses are presented in Tables 24-28, below. Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. The ALT and AST levels showed that the antisense compounds were well tolerated at all administered doses.

These results show improvement in potency for antisense compounds comprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1 conjugate and some phosphodiester linkages was as potent as ISIS 647535, which comprises the same conjugate and all internucleoside linkages within the ASO are phosphorothioate.

TABLE 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Dose SEQ (mg/ ED₅₀ 3′ Internucleoside ID ASO kg) % PBS (mg/kg) Conjugate linkage/Length No. PBS 0 99 — — — ISIS 1 104 13.2 None PS/20 821 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc ₃ -1 PS/20 822 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc ₃ -1 PS/PO/20 822 647536 1 60 3 31 10 21

TABLE 25 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice SEQ Dose % ED₅₀ 3′ Internucleoside ID ASO (mg/kg) PBS (mg/kg) Conjugate Linkage/Length No. PBS 0 99 — — — ISIS 1 104 23.2 None PS/20 821 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1 GalNAc ₃ -1 PS/20 822 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc ₃ -1 PS/PO/20 822 647536 1 60 3 31 10 21

TABLE 26 Effect of ASO treatment on triglyceride levels in transgenic mice SEQ Dose % ED₅₀ 3′ Internucleoside ID ASO (mg/kg) PBS (mg/kg) Conjugate Linkage/Length No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/20 821 304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc ₃ -1 PS/20 822 647535 1 70 3 34 10 23 ISIS 0.3 95 1.9 GalNAc ₃ -1 PS/PO/20 822 647536 1 66 3 31 10 23

TABLE 27 Effect of ASO treatment on total cholesterol levels in transgenic mice Dose 3′ Internucleoside ASO (mg/kg) % PBS Conjugate Linkage/Length SEQ ID No. PBS 0 96 — — ISIS 1 104 None PS/20 821 304801 3 96 10 86 30 72 ISIS 0.3 93 GalNAc ₃ -1 PS/20 822 647535 1 85 3 61 10 53 ISIS 0.3 115 GalNAc ₃ -1 PS/PO/20 822 647536 1 79 3 51 10 54

TABLE 28 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose SEQ (mg/ HDL LDL 3′ Internucleoside ID ASO kg) % PBS % PBS Conjugate Linkage/Length No. PBS 0 131 90 — — ISIS 1 130 72 None PS/20 821 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3 98 86 GalNAc ₃ -1 PS/20 822 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc ₃ -1 PS/PO/20 822 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency of an antisense compound. The results also show equal potency of a GalNAc₃-1 conjugated antisense compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six phosphodiester linkages) and a full phosphorothioate version of the same antisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisense compounds. For example, they resist nuclease digestion and they bind proteins resulting in accumulation of compound in the liver, rather than in the kidney/urine. These are desirable properties, particularly when treating an indication in the liver. However, phosphorothioate linkages have also been associated with an inflammatory response. Accordingly, reducing the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, but also lower concentration of the compound in liver, increase concentration in the kidney and urine, decrease stability in the presence of nucleases, and lower overall potency. The present results show that a GalNAc₃-1 conjugated antisense compound where certain phosphorothioate linkages have been replaced with phosphodiester linkages is as potent against a target in the liver as a counterpart having full phosphorothioate linkages. Such compounds are expected to be less proinflammatory (See Example 24 describing an experiment showing reduction of PS results in reduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1 In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17, were evaluated in a dose-dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNA levels. Further, the antisense compound comprising the GalNAc₃-1 conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 440762). These results demonstrate that the potency benefit of GalNAc₃-1 conjugates are observed using antisense oligonucleotides complementary to a different target and having different chemically modified nucleosides, in this instance modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar moiety).

TABLE 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice Liver SEQ Dose % ED₅₀ Internucleoside ID ASO (mg/kg) PBS (mg/kg) 3′ Conjugate linkage/Length No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/14 823 440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc ₃ -1 PS/14 824 651900 0.2 63 0.7 20 2 6 7 5

Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol

The hPBMC assay was performed using BD Vautainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A 60 μl sample was pipette into a sample vial (Beckman Coulter) with 600 μl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 μl/well of 96-well tissue culture plate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 μl/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay for GalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluated for proinflammatory effect in hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for IL-6 release in the assay. The hPBMCs were isolated from fresh, volunteered donors and were treated with ASOs at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hr treatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ and E_(max) was calculated using standard procedures. Results are expressed as the average ratio of E_(max)/EC₅₀ from two donors and is denoted as “E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in the proinflammatory response and the higher ratio indicates a relative increase in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compound was the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO, ISIS 647535 was slightly less proinflammatory than its non-conjugated counterpart ISIS 304801. These results indicate that incorporation of some PO linkages reduces proinflammatory reaction and addition of a GalNAc₃-1 conjugate does not make a compound more proinflammatory and may reduce proinflammatory response. Accordingly, one would expect that an antisense compound comprising both mixed PS/PO linkages and a GalNAc₃-1 conjugate would produce lower proinflammatory responses relative to full PS linked antisense compound with or without a GalNAc₃-1 conjugate. These results show that GalNAc₃-1 conjugated antisense compounds, particularly those having reduced PS content are less proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound, particularly one with reduced PS content, can be administered at a higher dose than a counterpart full PS antisense compound lacking a GalNAc₃-1 conjugate. Since half-life is not expected to be substantially different for these compounds, such higher administration would result in less frequent dosing. Indeed such administration could be even less frequent, because the GalNAc₃-1 conjugated compounds are more potent (See Examples 20-22) and re-dosing is necessary once the concentration of a compound has dropped below a desired level, where such desired level is based on potency.

TABLE 30 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISIS G_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds) TNFα 825 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 826 353512 G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC 821 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds) T_(es)T_(es)T_(es)A_(es)T_(e) III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC 822 647535 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - III GalNAc ₃ -1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC 821 616468 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) III

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “A_(do)′-GalNAc₃-1_(a)” indicates a conjugate having the structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of the antisense oligonucleotide, as indicated.

TABLE 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMC assay Inter- nucleoside SEQ EC₅₀ E_(max) E_(max)/ 3′ Linkage/ ID ASO (μM) (μM) EC₅₀ Conjugate Length No. ISIS 353512 0.01 265.9 26,590 None PS/20 826 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 821 ISIS 647535 0.12 138 1,150 GalNAc ₃ -1 PS/20 822 ISIS 616468 0.32 71.52 224 None PS/PO/20 821

Example 25: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting Human ApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells from transgenic mice at a density of 25,000 cells per well were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the hApoC III mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results are presented in Table 32. As illustrated, comparable potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.

TABLE 32 Modified ASO targeting human ApoC III in primary hepatocytes Internucleoside SEQ ASO IC₅₀ (μM) 3′ Conjugate linkage/Length ID No. ISIS 0.44 None PS/20 821 304801 ISIS 0.31 GalNAc ₃ -1 PS/20 822 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugation that are observed in vivo were not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates relative to oligonucleotides that lacking the GalNAc conjugate. (see Examples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or with PBS treated control once per week for two weeks. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III protein levels in the liver as described above (Example 20). Data from those analyses are presented in Table 33, below.

These results show reduction in potency for antisense compounds with PO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33 Effect of ASO treatment on ApoC III protein levels in human ApoC III transgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) % PBS Conjugate linkage/Length No. PBS 0 99 — — ISIS 25 mg/kg/wk 24 None Full PS 821 304801 for 2 wks ISIS 25 mg/kg/wk 40 None 14 PS/6 PO 821 616468 for 2 wks

Example 27: Compound 56

Compound 56 is commercially available from Glen Research or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2. Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.

Compound 57 is meant to be representative and not intended to be limiting as other mono-protected substituted or unsubstituted alkyl diols including but not limited to those presented in the specification herein can be used to prepare phosphoramidites having a predetermined composition.

Example 29: Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. J. Org. Chem., 2013, 3, 566-577; and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to those reported in scientific and patent literature by Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., published PCT International Application, WO 2004063208.

Example 30: Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

Compound 63c is prepared using procedures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 65 is prepared using procedures similar to those reported by Or et al., published PCT International Application, WO 2009003009. The protecting groups used for Compound 65 are meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 33: Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 68 is commercially available. The protecting group used for Compound 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 34: Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

Compound 76 was prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus Via Solid Support (Method I)

wherein GalNAc₃-2 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2 (GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56 and 60 were prepared as per the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks including but not limited those presented in the specification herein can be used to prepare an oligomeric compound having a phosphodiester linked conjugate group at the 5′ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 cluster phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example 35. This alternative method allows a one-step installation of the phosphodiester linked GalNAc₃-2 conjugate to the oligomeric compound at the final step of the synthesis. The phosphoramidites illustrated are meant to be representative and not intended to be limiting, as other phosphoramidite building blocks including but not limited to those presented in the specification herein can be used to prepare oligomeric compounds having a phosphodiester conjugate at the 5′ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 39: General Method for the Preparation of Oligomeric Compound 83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1 Modified for 5′ End Attachment) Via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4. Compounds 83a and 83b are commercially available. Oligomeric Compound 83e comprising a phosphodiester linked hexylamine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia provided the 5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3 (GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40: General Method for the Preparation of Oligomeric Compound 89 Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′ Terminus Via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4 (GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 is commercially available. Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are prepared as per the procedures illustrated in Examples 28 and 36. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a phosphodiester linked conjugate at the 3′ terminus with a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 41: General Method for the Preparation of ASOs Comprising a Phosphodiester Linked GalNAc₃-2 (See Example 37, Bx is Adenine) Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation of ISIS 661134)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and ^(m)C residues. Phosphoramidite compounds 56 and 60 were used to synthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered at a 4 fold excess over the initial loading of the solid support and phosphoramidite coupling was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing the dimethoxytrityl (DMT) groups from 5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during the coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 20% diethylamine in toluene (v/v) with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 6 h. The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

TABLE 34 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ position targeting SRB-1 ISIS Observed SEQ ID No. Sequence (5′ to 3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) 6482.2 6481.6 827 A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of GalNAc₃-2_(a) is shown in Example 37.

Example 42: General Method for the Preparation of ASOs Comprising a GalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises a GalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

TABLE 34a ASO comprising a GalNAc₃-3 conjugate at the 5′ position via a hexylamino phosphodiester linkage targeting Malat-1 ISIS Calcd Observed SEQ ID No. Sequence (5′ to 3′) Conjugate Mass Mass No. 661166 5′-GalNAc ₃ -3 _(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) 5′-GalNAc ₃ -3 8992.16 8990.51 828 ^(m)C_(ds)A_(ds)A_(ds)G_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “5′-GalNAc₃-3a” is shown in Example 39.

Example 43: Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (See Examples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 In Vivo

ISIS 661134 (see Example 41) comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9) were included in the study for comparison and are described previously in Table 17.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 661134 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

As illustrated in Table 35, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1 conjugate linked at the 3′ terminus (ISIS 651900) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was equipotent compared to ISIS 651900, which comprises the GalNAc₃-1 conjugate at the 3′ terminus.

TABLE 35 ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1 SRB-1 ISIS Dosage mRNA levels ED₅₀ SEQ ID No. (mg/kg) (% PBS) (mg/kg) Conjugate No. PBS 0 100 — — 440762 0.2 116 2.58 No conjugate 823 0.7 91 2 69 7 22 20 5 651900 0.07 95 0.26 3′ GalNAc ₃ -1 824 0.2 77 0.7 28 2 11 7 8 661134 0.07 107 0.25 5′ GalNAc ₃ -2 827 0.2 86 0.7 28 2 10 7 6

Structures for 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previously in Examples 9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same manner as illustrated in Example 20. Liver sample was minced and extracted using standard protocols. The full length metabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) were identified and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a detectable level, were observed. Unlike its counterpart, additional metabolites similar to those reported previously in Table 23a were observed for the ASO having the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 651900). These results suggest that having the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate may improve the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOs Comprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ Terminus Targeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′ terminus each targeting SRB-1 were tested in a single administration study for their ability to inhibit SRB-1 in mice. The parent unconjugated compound, ISIS 353382 was included in the study for comparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten 2′-deoxyribonucleosides and each wing region comprises five 2′-MOE modified nucleosides. The ASOs were prepared using similar methods as illustrated previously in Example 19 and are described Table 36, below.

TABLE 36 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminus targeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no 829 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) conjugate 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 830 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO 830 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) with GalNAc ₃ -1 conjugate

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “GalNAc₃-1” is shown in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 655862 or with PBS treated control. Each treatment group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides comprising the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showed substantial improvement in potency comparing to the unconjugated antisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixed PS/PO linkages showed an improvement in potency relative to full PS (ISIS 655861).

TABLE 37 Effect of PO/PS linkages on antisense inhibition of ASOs comprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISIS Dosage SRB-1 mRNA ED₅₀ SEQ ID No. (mg/kg) levels (% PBS) (mg/kg) Chemistry No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS without 829 (parent) 10 52.40 conjugate 30 24.95 655861 0.5 81.22 2.2 Full PS with 830 1.5 63.51 GalNAc₃-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3 Mixed PS/PO 830 1.5 45.78 with GalNAc₃-1 5 19.70 conjugate 15 12.90

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Organ weights were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 38) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS control. Further, the ASO with mixed PS/PO linkages (ISIS 655862) showed similar transaminase levels compared to full PS (ISIS 655861).

TABLE 38 Effect of PO/PS linkages on transaminase levels of ASOs comprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISIS Dosage ALT AST No. (mg/kg) (U/L) (U/L) Chemistry SEQ ID No. PBS 0 28.5 65 — 353382 3 50.25 89 Full PS without 829 (parent) 10 27.5 79.3 conjugate 30 27.3 97 655861 0.5 28 55.7 Full PS with 830 1.5 30 78 GalNAc ₃ -1 5 29 63.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 830 1.5 21.7 58.5 GalNAc ₃ -1 5 29.3 69 15 22 61

Example 45: Preparation of PFP Ester, Compound 110a

Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b (38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves in dichloromethane (200 mL), and stirred for 16 hours at room temperature. At that time, the organic layer was filtered thru celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced under reduced pressure. The resultant oil was purified by silica gel chromatography (2%->10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was consistent with the structure.

Compounds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 105a and 105b in >90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90 under the same conditions as for compounds 901a-d, to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 106a and 106b were treated to the same conditions as for compounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as for compounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 109a and 109b in >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was consistent with the structure. Alternatively, Compound 110b can be prepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5′-hexylamino modified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃ cluster dissolved in DMSO (50 μL) was added. If the PFP ester precipitated upon addition to the ASO solution DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h of mixing at room temperature. The resulting solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was then lyophilized to dryness and redissolved in concentrated aqueous ammonia and mixed at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to provide the GalNAc₃ conjugated oligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10 (GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 μmol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 μL) to fully dissolve the PFP ester. The reaction was complete after 16 h of mixing at room temperature. The solution was diluted with water to 12 mL total volume and spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous ammonia with mixing at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7 μmol).

GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′) 5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o) Hexylamine 831 A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) GalNAc ₃ -10 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added, along with N,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction was allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%->10% methanol/dichloromethane) to give compounds 92a and 92b in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL of dichloromethane and 20 mL of trifluoroacetic acid at room temperature for 16 hours. The resultant solution was evaporated and then dissolved in methanol and treated with DOWEX-OH resin for 30 minutes. The resultant solution was filtered and reduced to an oil under reduced pressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7 g, 9.6 mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 mmoles), and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%->20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated over Raney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalyst was removed by filtration thru celite, and the ethanol removed under reduced pressure to give compounds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53 mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30 mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%->20% methanolldichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent with the structure.

Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76 mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50 mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%->20% methanol/dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10% Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At that time, the catalyst was removed by filtration thru celite, and the organics removed under reduced pressure to give compounds 101 a-d in 80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%->5% methanol/dichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate (22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite and washed the pad with 1:1 methanol/ethyl acetate. The filtrate and the washings were combined and concentrated to dryness to yield Compound 105a (quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction was stirred at room temperature for 6 h. Solvent was removed under reduced pressure to get an oil. The residue was dissolved in CH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100 mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20% MeOH in dichloromethane to yield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethyl acetate (4 mL/4 mL).

Palladium on carbon (wet, 0.14 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added and the reaction was stirred at room temperature for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in CH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solution and brine and dried over anhydrous Na₂SO₄ and filtered. The organic layer was concentrated to dryness and the residue obtained was purified by silica gel column chromatography and eluted with 3 to 15% MeOH in dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and ¹H NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl acetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHCO₃ solution. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na₂SO₄. The dichloromethane solution was concentrated to dryness and purified with silica gel column chromatography and eluted with 5 to 10% MeOH in dichloromethane to yield compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 min. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added and the reaction mixture was stirred for 18 h under an argon atmosphere. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf=0.47). The solvent was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150 mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL). Organic layer was dried with Na₂SO₄. Drying agent was removed by filtration and organic layer was concentrated by rotary evaporation. Crude mixture was purified by silica gel column chromatography and eluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g, 78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Mass m/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled solution of Compound 122 (7.75 g, 13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The remaining solution was cooled an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon standing overnight. The organic layer was separated dried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. Product was used without any further purification. M.W.cal:574.36; M.W.fd:575.3 [M+H]⁺.

Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) and the resulting reaction mixture was cooled in an ice bath. To this N,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere. The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was completed after 1 h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1 M NaHSO₄ (3×100 mL), aqueous saturated NaHCO₃ (3×100 mL) and brine (2×100 mL). The organic phase separated dried (Na₂SO₄), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹H NMR were consistent with structure. Mass m/z 778.4 [M+H]⁺.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room temperature for 1 h. The reaction mixture was co-evaporated with toluene (30 mL) under reduced pressure to dryness. The residue obtained was co-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128 (1.67 g) as trifluoro acetate salt and used for next step without further purification. LCMS and ¹H NMR were consistent with structure. Mass m/z 478.2 [M+H]⁺.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅ under reduced pressure in a round bottom flask and then dissolved in anhydrous DMF (1 mL) and stirred for 5 min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture was stirred at room temperature under an argon atmosphere. The reaction was complete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO₄ (3×20 mL), aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organic phase was separated, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography using 5-15% MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and ¹H NMR are consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20 mL scintillation vial. To this was added a small amount of 10% Pd/C (0.015 mg) and the reaction vessel was flushed with H₂ gas. The reaction mixture was stirred at room temperature under H₂ atmosphere for 18 h. The reaction mixture was filtered through a pad of Celite and the Celite pad was washed with methanol. The filtrate washings were pooled together and concentrated under reduced pressure to yield Compound 130 (0.08 g). LCMS and ¹H NMR were consistent with structure. The product was used without further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To this solution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 μL) drop wise with stirring. The reaction was completed after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHCl₃ (˜10 mL). The organic layer was partitioned against NaHSO₄ (1 M, 10 mL), aqueous saturated NaHCO₃ (10 mL) and brine (10 mL) three times each. The organic phase separated and dried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without further purification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

Synthesis of Compound 134. To a Merrifield flask was added aminomethyl VIMAD resin (2.5 g, 450 μmol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was swelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100 mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solution was allowed to stir for 5 min and was then added to the Merrifield flask with shaking. The suspension was allowed to shake for 3 h. The reaction mixture was drained and the resin was washed with acetonitrile, DMF and DCM. New resin loading was quantitated by measuring the absorbance of the DMT cation at 500 nm (extinction coefficient=76000) in DCM and determined to be 238 μmol/g. The resin was capped by suspending in an acetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterative Fmoc-based solid phase peptide synthesis methods. A small amount of solid support was withdrawn and suspended in aqueous ammonia (28-30 wt %) for 6 h. The cleaved compound was analyzed by LC-MS and the observed mass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phase peptide synthesis methods.

The solid support bound compound 143 was synthesized using standard solid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled and the solid support was filtered. The filtrate was concentrated and the residue dissolved in water and purified by HPLC on a strong anion exchange column. The fractions containing full length compound 144 were pooled together and desalted. The resulting GalNAc₄-11 conjugated oligomeric compound was analyzed by LC-MS and the observed mass was consistent with structure.

The GalNAc₄ cluster portion of the conjugate group GalNAc₄-11 (GalNAc₄-11_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-11_(a)-CM) is shown below:

Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was quenched by pouring into solution of saturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organic layer was separated, washed with brine, dried over MgSO₄, filtered, and was concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂ to yield Compound 112 (16.53 g, 63%). LCMS and ¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in CH₂Cl₂, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL). N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15 minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added and stirred for 2 h under nitrogen atmosphere. The reaction was diluted with EtOAc and washed with saturated aqueous NaHCO₃ and brine. The organics phase was separated, dried (MgSO₄), filtered, and concentrated to an orange syrup. The crude material was purified by column chromatography 2-5% MeOH in CH₂Cl₂ to yield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent with the expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (350 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with desired product. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The reaction mixture was allowed to stir for 15 minutes at room temperature under nitrogen. A solution of compound 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of the reaction was adjusted to pH=9-10 by addition of DIEA (if necessary). The reaction was allowed to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with EtOAc (100 mL), and washed with aqueous saturated aqueous NaHCO₃, followed by brine. The organic phase was separated, dried over MgSO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂ to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR were consistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the desired product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76 mmol) were added. The reaction mixture turned magenta upon contact, and gradually turned orange over the next 30 minutes. Progress of reaction was monitored by TLC and LCMS. Upon completion (formation of the PFP ester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction was adjusted to pH=9-10 by addition of N,N-Diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for ˜30 min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. The organic phase separated, dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g, 55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The reaction mixture was purged by bubbling a stream of argon thru the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (35 mg). Hydrogen gas was bubbled thru the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 153 (0.33 g, quantitative). The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL) with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL, 0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reaction mixture was stirred under nitrogen for ˜30 min. The reaction mixture turned magenta upon contact, and gradually turned orange. The pH of the reaction mixture was maintained at pH=9-10 by adding more N,-Diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), and washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10% MeOH in CH₂Cl₂ to yield Compound 154 (0.29 g, 79%). LCMS and ¹H NMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL). Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbon powder, wet) was added. The reaction mixture was stirred at room temperature under hydrogen for 18 h. The reaction mixture was filtered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined filtrate was washed and concentrated to dryness. The residue was purified by silica gel column chromatography and eluted with 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL). HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81 mmol) were added and the reaction mixture was stirred at room temperature for 5 minutes. To this a solution of compound 47 (2.96 g, 7.04 mmol) was added. The reaction was stirred at room temperature for 8 h. The reaction mixture was poured into a saturated NaHCO₃ aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na₂SO₄), filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25 g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reduced pressure. The dried compound was dissolved in anhydrous DMF (50 mL). To this 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL, 3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite (3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred t under an argon atmosphere for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL). The reaction mixture was washed with saturated NaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90% ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and ³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, was prepared using standard oligonucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous ammonia yielded compound 160. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9 (GalNAc₃-9_(a)-CM) is shown below:

Example 53: Alternate Procedure for Preparation of Compound 18 (GalNAc₃—La and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Boc protected diamino propane (1 eq) to provide alcohol 162a or 162b. When unprotected propanediamine was used for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected using CbzCl to provide 162b as a white solid after purification by column chromatography. Alcohol 162b was further reacted with compound 4 in the presence of TMSOTf to provide 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine (3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18. The above method greatly facilitates purification of intermediates and minimizes the formation of byproducts which are formed using the procedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18 (GalNAc₃—La and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedure outlined in example 53 above and reacted with mono-Boc protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or trifluoroacetic acid to provide the triamine which was reacted with the PFP activated acid 166 in the presence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the corresponding acid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF. The precursor acid in turn was prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by reaction with 1,6-hexanediol (or 1,5-pentanediol or other diol for other n values) (2-4 eq) and TMSOTf using conditions described previously in example 47.

Example 55: Dose-Dependent Study of Oligonucleotides Comprising Either a 3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃ conjugate groups was attached at either the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 39 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) Motif Conjugate ID No. ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 none 829 353382 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) (parent) ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 830 655861 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 830 664078 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A_(do′) -GalNAc ₃ -9 _(a) ISIS GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 831 661161 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 831 665001 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)c indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-9 was shown previously in Example 52. The structure of GalNAc₃-3 was shown previously in Example 39. The structure of GalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664078, 661161, 665001 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 and ISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate at the 3′ terminus was essentially equipotent compared to ISIS 655861, which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′ conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potency compared to the 3′ conjugated antisense oligonucleotides (ISIS 655861 and ISIS 664078).

TABLE 40 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 SRB-1 Dosage mRNA (% ISIS No. (mg/kg) Saline) Conjugate Saline n/a 100 353382 3 88 none 10 68 30 36 655861 0.5 98 GalNac ₃ -1 (3′) 1.5 76 5 31 15 20 664078 0.5 88 GalNac ₃ -9 (3′) 1.5 85 5 46 15 20 661161 0.5 92 GalNac ₃ -3 (5′) 1.5 59 5 19 15 11 665001 0.5 100 GalNac ₃ -8 (5′) 1.5 73 5 29 15 13

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 41 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.2 30 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNac ₃ -1 (3′) 1.5 23 48 0.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3 GalNac ₃ -9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2 661161 0.5 101 162 0.1 34.17 GalNac ₃ -3 (5′) 1.5 g 42 100 0.1 33.37 5 g 23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32 GalNac ₃ -8 (5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56: Dose-Dependent Study of Oligonucleotides Comprising Either a 3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety) except for ISIS 655861 which had the GalNAc₃ conjugate group attached at the 3′ terminus.

TABLE 42 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) Motif Conjugate ID No. ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 no conjugate 829 353382 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) (parent) ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 830 655861 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS GalNAc ₃ -2 _(a) - _(o′) 5/10/5 GalNAc ₃ -2 831 664507 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 831 661161 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -5 _(a) - _(o′) 5/10/5 GalNAc ₃ -5 831 666224 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -6 _(a) - _(o′) 5/10/5 GalNAc ₃ -6 831 666961 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -7 _(a) - _(o′) 5/10/5 GalNAc ₃ -7 831 666981 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -10 _(a) - _(o′) 5/10/5 GalNAc ₃ -10 831 666881 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)c indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-2_(a) was shown previously in Example 37. The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-5_(a) was shown previously in Example 49. The structure of GalNAc₃-6_(a) was shown previously in Example 51. The structure of GalNAc₃-7_(a) was shown previously in Example 48. The structure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the conjugated antisense oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). The 5′ conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3′ conjugated antisense oligonucleotide.

TABLE 43 SRB-1 Dosage mRNA (% ISIS No. (mg/kg) Saline) Conjugate Saline n/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNac ₃ -1 (3′) 1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNac ₃ -2 (5′) 1.5 73.2 5 31.3 15 10.8 661161 0.5 90.7 GalNac ₃ -3 (5′) 1.5 67.6 5 24.3 15 11.5 666224 0.5 96.1 GalNac ₃ -5 (5′) 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5 GalNAc ₃ -6 (5′) 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc ₃ -7 (5′) 1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0 GalNAc ₃ -10 (5′) 1.5 65.8 5 26.0 15 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.

TABLE 44 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 54 0.1 28 655861 0.5 25 71 0.2 34 GalNac ₃ -1 (3′) 1.5 28 60 0.2 26 5 26 63 0.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNac ₃ -2 (5′) 1.5 24 49 0.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac ₃ -3 (5′) 1.5 g 37 74 0.2 25 5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5 34 48 0.2 21 GalNac ₃ -5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 32 49 0.1 26 666961 0.5 17 63 0.2 26 GalNAc ₃ -6 (5′) 1.5 23 68 0.2 26 5 25 66 0.2 26 15 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc ₃ -7 (5′) 1.5 30 55 0.2 24 5 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27 GalNAc ₃ -10 1.5 23 59 0.2 24 (5′) 5 45 70 0.2 26 15 21 57 0.2 24

Example 57: Duration of Action Study of Oligonucleotides Comprising a 3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitored over the course of 42 days for ApoC-III and plasma triglycerides (Plasma TG) levels. The study was performed using 3 transgenic mice that express human APOC-III in each group.

TABLE 45 Modified ASO targeting ApoC III SEQ ASO Sequence (5′ to 3′) Linkages ID No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 821 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds) PS 822 647535 A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ - 1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds) PO/PS 822 647536 A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ - 1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

TABLE 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (% Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline  0 mg/kg ApoC-III 98 100 100 95 116 ISIS 304801 30 mg/kg ApoC-III 28 30 41 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10 mg/kg ApoC-III 18 16 17 35 51 Saline  0 mg/kg Plasma TG 121 130 123 105 109 ISIS 304801 30 mg/kg Plasma TG 34 37 50 69 69 ISIS 647535 10 mg/kg Plasma TG 18 14 24 18 71 ISIS 647536 10 mg/kg Plasma TG 21 19 15 32 35

As can be seen in the table above the duration of action increased with addition of the 3′-conjugate group compared to the unconjugated oligonucleotide. There was a further increase in the duration of action for the conjugated mixed PO/PS oligonucleotide 647536 as compared to the conjugated full PS oligonucleotide 647535.

Example 58: Dose-Dependent Study of Oligonucleotides Comprising a 3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc₄-11) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 663748 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-1 and GalNAc₄-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). The two conjugated oligonucleotides, GalNAc₃-1 and GalNAc₄-11 were equipotent.

TABLE 47 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence (5′ to 3′) Dose mg/kg control No. Saline 100 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.6 73.45 823 440762 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 62.75 824 651900 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1 _(a) 0.6 29.14 2 8.61 6 5.62 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 63.99 824 663748 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₄ -11 _(a) 0.6 33.53 2 7.58 6 5.52

Capital letters indicate the nucleobase for each nucleoside and ^(m)c indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 48 below.

TABLE 48 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 48 0.1 39 651900 0.2 32 115 0.2 39 GalNac ₃ -1 (3′) 0.6 33 61 0.1 35 2 30 50 0.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNac ₄ -11 0.6 34 60 0.1 35 (3′) 2 44 62 0.1 36 6 38 71 0.1 33

Example 59: Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of FXI in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugate groups was attached at the 3′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 49 Modified ASOs targeting FXI SEQ Link- ID ASO Sequence (5′ to 3′) ages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 832 404071 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 833 656172 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do′) - GalNAc ₃ -1 _(a) ISIS T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PO/PS 833 656173 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do′) - GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)c indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously twice a week for 3 weeks at the dosage shown below with ISIS 404071, 656172, 656173 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Plasma FXI protein levels were also measured using ELISA. FXI mRNA levels were determined relative to total RNA (using RIBOGREEN®), prior to normalization to PBS-treated control. The results below are presented as the average percent of FXI mRNA levels for each treatment group. The data was normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

TABLE 50 Factor XI mRNA (% Saline) Dose ASO mg/kg % Control Conjugate Linkages Saline 100 none ISIS 3 92 none PS 404071 10 40 30 15 ISIS 0.7 74 GalNAc ₃ -1 PS 656172 2 33 6 9 ISIS 0.7 49 GalNAc ₃ -1 PO/PS 656173 2 22 6 1

As illustrated in Table 50, treatment with antisense oligonucleotides lowered FXI mRNA levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a Factor XI protein (% Saline) Dose Protein (% ASO mg/kg Control) Conjugate Linkages Saline 100 none ISIS 3 127 none PS 404071 10 32 30 3 ISIS 0.7 70 GalNAc ₃ -1 PS 656172 2 23 6 1 ISIS 0.7 45 GalNAc ₃ -1 PO/PS 656173 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 51 Dosage Total Total ISIS No. mg/kg ALT AST Albumin Bilirubin CRE BUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.1 0.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.2 0.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNac ₃ -1 2 33.0 51.8 2.9 0.2 0.2 22.0 (3′) 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.5 3.0 0.2 0.2 24.9 GalNac ₃ -1 2 85.8 71.5 3.2 0.2 0.2 21.0 (3′) 6 114.0 101.8 3.3 0.2 0.2 22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 52 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′) Motif Conjugate No. ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 none 829 353382 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 830 655861 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1a ISIS G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 830 655862 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1a ISIS GalNAc ₃ -3 _(a-o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -3 831 661161 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -8 _(a-o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -8 831 665001 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 830 664078 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -9 _(a) ISIS GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -6 831 666961 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -2 _(a) - _(o′) 5/10/5 GalNAc ₃ -2 831 664507 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -10 _(a) - _(o′) 5/10/5 GalNAc ₃ -10 831 666881 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -5 _(a) - _(o′) 5/10/5 GalNAc ₃ -5 831 666224 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS GalNAc ₃ -7 _(a) - _(o′) 5/10/5 GalNAc ₃ -7 831 666981 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)c indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-3a was shown previously in Example 39. The structure of GalNAc₃-8a was shown previously in Example 47. The structure of GalNAc₃-9a was shown previously in Example 52. The structure of GalNAc₃-6a was shown previously in Example 51. The structure of GalNAc₃-2a was shown previously in Example 37. The structure of GalNAc₃-10a was shown previously in Example 46. The structure of GalNAc₃-5a was shown previously in Example 49. The structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modified oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the SRB-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results are presented in Table 53.

The results show that, under free uptake conditions in which no reagents or electroporation techniques are used to artificially promote entry of the oligonucleotides into cells, the oligonucleotides comprising a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.

TABLE 53 Internucleoside SEQ ID ASO IC₅₀ (nM) linkages Conjugate No. ISIS 190^(a ) PS none 829 353382 ISIS  11^(a) PS GalNAc ₃ -1 830 655861 ISIS  3 PO/PS GalNAc ₃ -1 830 655862 ISIS  15^(a) PS GalNAc ₃ -3 831 661161 ISIS 20 PS GalNAc ₃ -8 831 665001 ISIS 55 PS GalNAc ₃ -9 830 664078 ISIS  22^(a) PS GalNAc ₃ -6 831 666961 ISIS 30 PS GalNAc ₃ -2 831 664507 ISIS 30 PS GalNAc ₃ -10 831 666881 ISIS  30^(a) PS GalNAc ₃ -5 831 666224 ISIS 40 PS GalNAc ₃ -7 831 666981 ^(a)Average of multiple runs.

Example 61: Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared by addition of benzyl (perfluorophenyl) glutarate to compound 171. The benzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound 175, comprising a GalNAc₃-12 conjugate group, was prepared from compound 174 using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12 (GalNAc₃-12_(a)-CM-) is shown below:

Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example 2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, was prepared from compound 177 using the general procedures illustrated in Example 49. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-13 (GalNAc₃-13_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a GalNAc₃-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-14 (GalNAc₃-14_(a)-CM-) is shown below:

Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared using the general procedure shown in Example 31. Oligomeric compound 197, comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194 and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15 (GalNAc₃-15_(a)-CM-) is shown below:

Example 65: Dose-Dependent Study of Oligonucleotides Comprising a 5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54 Modified ASOs targeting SRB-1 SEQ ISIS No. Sequences (5′ to 3′) Conjugate ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) none 829 T_(e) 661161 GalNAc ₃ -3 _(a) - _(o′) GalNAc₃-3 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671144 GalNAc ₃ -12 _(a) - _(o′) GalNAc₃-12 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670061 GalNAc ₃ -13 _(a) - _(o′) GalNAc₃-13 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671261 GalNAc ₃ -14 _(a) - _(o′) GalNAc₃-14 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671262 GalNAc ₃ -15 _(a) - _(o′) GalNAc₃-15 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)c indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-12a was shown previously in Example 61. The structure of GalNAc₃-13a was shown previously in Example 62. The structure of GalNAc₃-14a was shown previously in Example 63. The structure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once or twice at the dosage shown below with ISIS 353382, 661161, 671144, 670061, 671261, 671262, or with saline. Mice that were dosed twice received the second dose three days after the first dose. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. No significant differences in target knockdown were observed between animals that received a single dose and animals that received two doses (see ISIS 353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5 mg/kg). The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

TABLE 55 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA ED₅₀ No. (mg/kg) (% Saline) (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.0 22.4 none 10 69.2 30 34.2 2 × 15  36.0 661161 0.5 87.4 2.2 GalNAc₃-3 1.5 59.0 5 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.5 76.1 5 32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 15 13.3 671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5 109.4 9.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56 Total Dosage ALT AST Bilirubin BUN ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.2 36 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15 22 59 0.2 33 661161 0.5 39 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.2 28 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃- 1.5 26 55 0.2 28 12 5 48 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃- 1.5 24 45 0.2 35 13 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33 GalNAc₃- 1.5 34 62 0.1 33 14 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 24 51 0.2 29 GalNAc₃- 1.5 32 62 0.1 31 15 5 30 76 0.2 32 15 31 64 0.1 32

Example 66: Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃ Cluster

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM))

TABLE 57 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670690 GalNAc ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d) 834 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 670700 GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 670701 GalNAc ₃ -3 _(a) - _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 834 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 671165 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising various cleavable moieties all showed similar potencies.

TABLE 58 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8 GalNAc₃-3a A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3a T_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a A_(e) 1.5 63.3 5 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a T_(e) 1.5 59.2 5 35.8 15 17.7 671165 0.5 76.4 GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 59 Total BUN ISIS Dosage ALT AST Bilirubin (mg/ GalNAc₃ No. (mg/kg) (U/L) (U/L) (mg/dL) dL) Cluster CM Saline n/a 24 64 0.2 31 n/a n/a 661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 55 0.2 28 15 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 33 58 0.2 32 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27 GalNAc₃-3a A_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 670701 0.5 30 77 0.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 31 82 0.2 24 671165 0.5 44 84 0.2 26 GalNAc₃- A_(d) 1.5 47 71 0.1 24 13a 5 33 91 0.2 26 15 33 56 0.2 29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16 (GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-16 (GalNAc₃-16_(a)-CM-) is shown below:

Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-17 (GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-17 (GalNAc₃-17_(a)-CM-) is shown below:

Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-18 (GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-18 (GalNAc₃-18_(a)-CM-) is shown below:

Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, was prepared from compound 64 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which was prepared by adding triflic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80° C., then lowered to rt. Oligomeric compound 210, comprising a GalNAc₃-20 conjugate group, was prepared from compound 208 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-20 (GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20 (GalNAc₃-20_(a)-CM-) is shown below:

Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215, comprising a GalNAc₃-21 conjugate group, was prepared from compound 213 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21 (GalNAc₃-21_(a)-CM-) is shown below:

Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugate group, is prepared from compound 220 using the general procedure illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74: Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide.

TABLE 60 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) n/a n/a 829 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 829 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675441 GalNAc ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(dsTds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675442 GalNAc ₃ -18 _(a)-o′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

In all tables, capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-17a was shown previously in Example 68, and the structure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 60 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc conjugate showed similar potencies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 61 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38 n/a n/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a A_(d) 1.5 75.96 5 30.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a PO 1.5 57.88 5 21.22 15 16.49 675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49 675442 0.5 95.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 62 below.

TABLE 62 Total BUN ISIS Dosage ALT AST Bilirubin (mg/ GalNAc₃ No. (mg/kg) (U/L) (U/L) (mg/dL) dL) Cluster CM Saline n/a 26 59 0.16 42 n/a n/a 353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34 661161 0.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.15 39 15 32 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 48 0.12 32 5 19 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36 GalNAc₃- A_(d) 1.5 60 95 0.16 34 17a 5 27 75 0.14 37 15 24 61 0.14 36 675442 0.5 26 65 0.15 37 GalNAc₃- A_(d) 1.5 25 64 0.15 43 18a 5 27 69 0.15 37 15 30 84 0.14 37

Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising a 5′-Conjugate Group

The PK of the ASOs in Tables 54, 57 and 60 above was evaluated using liver samples that were obtained following the treatment procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using standard protocols and analyzed by IP-HPLC-MS alongside an internal standard. The combined tissue level (μg/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate (“parent,” which is Isis No. 353382 in this case) was measured using the appropriate extracted ion chromatograms (EIC).

TABLE 63 PK Analysis in Liver Parent Total Tissue ASO Tissue ISIS Dosage Level by UV Level by EIC GalNAc₃ No. (mg/kg) (μg/g) (μg/g) Cluster CM 353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7 GalNAc₃-3a A_(d) 15 63.2 44.1 671144 5 20.5 19.2 GalNAc₃-12a A_(d) 15 48.6 41.5 670061 5 31.6 28.0 GalNAc₃-13a A_(d) 15 67.6 55.5 671261 5 19.8 16.8 GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4 GalNAc₃-15a A_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a T_(d) 15 31.5 22.5 670700 5 19.3 10.9 GalNAc₃-3a A_(e) 15 38.1 20.0 670701 5 21.8 8.8 GalNAc₃-3a T_(e) 15 35.2 16.1 671165 5 27.1 26.5 GalNAc₃-13a A_(d) 15 48.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a PO 15 52.6 37.6 675441 5 25.4 19.0 GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7 GalNAc₃-18a A_(d) 15 39.6 29.0

The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a GalNAc₃ conjugate group than of the parent oligonucleotide that does not comprise a GalNAc₃ conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking into consideration the differences in dosing between the oligonucleotides with and without a GalNAc₃ conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a GalNAc₃ conjugate group was metabolized to the parent compound, indicating that the GalNAc₃ conjugate groups were cleaved from the oligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500 mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in EtOAc and washed with water, sat. NaHCO₃, brine, and dried over Na₂SO₄. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with EtOAc/hexanes (1:1) followed by 10% methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMR were consistent with the structure. 10 g (32.86 mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17 hours. The reaction mixture was then poured onto water, and extracted with EtOAc. The organic layer was washed with water three times and dried over Na₂SO₄. The organic layer was concentrated to dryness to give 5.3 g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4A molecular sieves (5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) under an inert atmosphere. After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat. NaHCO₃, water, brine, and dried over Na₂SO₄. The organic layer was concentrated to dryness and purified by column chromatography, eluted with a gradient of 2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMR were consistent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman's catalyst. After 8 hours, the catalyst was removed by filtration and the solvents removed to dryness to give compound 226. LCMS and NMR were consistent with the structure.

In order to generate compound 227, a solution of nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base (10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise with pentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the reaction was poured onto ice water and extracted with EtOAc. The organic layer was washed with water, brine, and dried over Na₂SO₄. The organic layer was concentrated to dryness and then recrystallized from heptane to give compound 227 as a white solid. LCMS and NMR were consistent with the structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 mL) for 2 hours. The reaction was then evaporated to dryness and purified by column chromatography, eluting with a gradient of 2 to 10% methanol in dichloromethane to give compound 228. LCMS and NMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in an atmosphere of hydrogen. After 12 hours, the catalyst was removed by filtration and the organic layer was evaporated to a solid that was used directly in the next step. LCMS and NMR were consistent with the structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 μl, 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removed under reduced pressure at 65° C. to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. NaHCO₃, brine, and dried over Na₂SO₄. After evaporation of the organic layer, the compound was purified by column chromatography and eluted with a gradient of 2 to 20% methanol in dichloromethane to give the coupled product. LCMS and NMR were consistent with the structure. The benzyl ester was deprotected with Pearlman's catalyst under a hydrogen atmosphere for 1 hour. The catalyst was them removed by filtration and the solvents removed to dryness to give the acid. LCMS and NMR were consistent with the structure. The acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was added and the reaction was purged with argon. Pentaflourotriflouro acetate (46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The color of the reaction changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The reaction was allowed to stir at room temperature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced pressure (rotovap) at 70° C. The residue was diluted with DCM and washed with 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. The organics were dried over Na₂SO₄, filtered, and were concentrated to dryness to give 225 mg of compound 229 as a brittle yellow foam. LCMS and NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, was prepared from compound 229 using the general procedure illustrated in Example 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugate group (GalNAc₃-23_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc₃-23 (GalNAc₃-23_(a)-CM) is shown below:

Example 77: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 64 Modified ASOs targeting SRB-1 GalNAc₃ SEQ ISIS No. Sequences (5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a)-_(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 829 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 673502 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 677844 GalNAc ₃ -9 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 677843 GalNAc ₃ -23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d) 831 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-1a A_(d) 830 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-19a A_(d) 830 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-20a A_(d) 830 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a)

The structure of GalNAc₃-1a was shown previously in Example 9, GalNAc₃-3a was shown in Example 39, GalNAc₃-9a was shown in Example 52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown in Example 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a) was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 64 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 89.18 GalNAc₃-3a A_(d) 1.5 77.02 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3a PO 1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75 GalNAc₃-10a A_(d) 1.5 41.05 5 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a A_(d) 1.5 93.04 5 40.77 15 16.95 677843 0.5 102.28 GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 15 13.26 655861 0.5 79.72 GalNAc₃-1a A_(d) 1.5 55.48 5 26.99 15 17.58 677841 0.5 67.43 GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 677842 0.5 64.13 GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN were also evaluated. Changes in body weights were evaluated, with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.

TABLE 66 Total BUN ISIS Dosage ALT AST Bilirubin (mg/ GalNAc₃ No. (mg/kg) (U/L) (U/L) (mg/dL) dL) Cluster CM Saline n/a 21 45 0.13 34 n/a n/a 661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 59 0.13 37 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 26 68 0.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34 GalNAc₃- A_(d) 1.5 20 46 0.17 32 10a 5 24 45 0.12 31 15 24 47 0.13 34 677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.13 35 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃- A_(d) 1.5 25 54 0.13 34 23a 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33 GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30 677841 0.5 32 54 0.13 34 GalNAc₃- A_(d) 1.5 24 56 0.14 34 19a 5 23 92 0.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃- A_(d) 1.5 24 57 0.14 34 20a 5 41 62 0.15 35 15 24 37 0.14 32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides Targeting Angiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.

TABLE 67 Modified ASOs targeting AGT ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 552668 ^(m)C_(es)A_(es) ^(m)C_(es)T_(es) n/a n/a 835 G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds) G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) A_(es)G_(es)G_(es)A_(es)T_(e) 669509 ^(m)C_(es)A_(es) ^(m)C_(es) GalNAc₃-1_(a) A_(d) 836 T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds) T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m) C_(ds) ^(m)C_(ds)A_(es)G_(es)G_(es) A_(es)T_(eo) A _(do′) -GalNAc ₃-1_(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 67 or with PBS. Each treatment group consisted of 4 animals. The rats were sacrificed 72 hours following the final dose. AGT liver mRNA levels were measured using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. AGT plasma protein levels were measured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBL International, Toronto, ON) with plasma diluted 1:20,000. The results below are presented as the average percent of AGT mRNA levels in liver or AGT protein levels in plasma for each treatment group, normalized to the PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotides lowered AGT liver mRNA and plasma protein levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 68 AGT liver mRNA and plasma protein levels AGT liver AGT plasma ISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 8 11 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in plasma and body weights were also measured at time of sacrifice using standard protocols. The results are shown in Table 69 below.

TABLE 69 Liver transaminase levels and rat body weights Body Dosage ALT AST Weight (% GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) of baseline) Cluster CM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93 194 30 59 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃- A_(d) 1 51 93 192 1a 3 48 85 189 10 56 95 189

Example 79: Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action in mice.

TABLE 70 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 821 T_(es)A_(es)T_(e) 647535 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1a A_(d) 822 T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663083 GalNAc ₃-3 _(a)-_(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 837 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449 GalNAc ₃-7 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 837 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 837 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451 GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 837 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six to eight week old transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 70 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results below are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels, showing that the oligonucleotides comprising a GalNAc conjugate group exhibited a longer duration of action than the parent oligonucleotide without a conjugate group (ISIS 304801) even though the dosage of the parent was three times the dosage of the oligonucleotides comprising a GalNAc conjugate group.

TABLE 71 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point (days APOC-III ISIS Dosage post- Triglycerides protein (% GalNAc₃ No. (mg/kg) dose) (% baseline) baseline) Cluster CM PBS n/a 3 97 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 42 91 105 304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 68 70 42 75 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 41 41 28 42 62 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 28 23 14 25 27 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26 GalNAc₃-7a A_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99 674450 10 3 33 30 GalNAc₃- A_(d) 7 35 34 10a 14 31 34 21 44 44 28 56 61 35 68 70 42 83 95 674451 10 3 35 33 GalNAc₃- A_(d) 7 24 32 13a 14 40 34 21 48 48 28 54 67 35 65 75 42 74 97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides Targeting Alpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 below were tested in a study for dose-dependent inhibition of A1AT in mice.

TABLE 72 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 838 G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1a A_(d) 839 G_(es)G_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 678381 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds) GalNAc₃-3a A_(d) 840 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678382 GalNAc ₃ -7 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds) GalNAc₃-7a A_(d) 840 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678383 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃- A_(d) 840 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 10a 678384 GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃- A_(d) 840 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 13a

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. A1AT liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. A1AT plasma protein levels were determined using the Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco, Salem, N.H.). The results below are presented as the average percent of A1AT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.

As illustrated in Table 73, treatment with antisense oligonucleotides lowered A1AT liver mRNA and A1AT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 476366).

TABLE 73 A1AT liver mRNA and plasma protein levels A1AT liver A1AT plasma ISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 30 38 656326 0.6 99 90 GalNAc₃-1a A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6 105 90 GalNAc₃-3a A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79 GalNAc₃-7a A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc₃-10a A_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc₃-13a A_(d) 2 65 59 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time of sacrifice using standard protocols. Body weights and organ weights were also measured. The results are shown in Table 74 below. Body weight is shown as % relative to baseline. Organ weights are shown as % of body weight relative to the PBS control group.

TABLE 74 Liver Kidney Body weight weight Spleen ISIS Dosage ALT AST BUN weight (% (Rel % (Rel % weight No. (mg/kg) (U/L) (U/L) (mg/dL) baseline) BW) BW) (Rel % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 34 68 35 116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123 656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 32 67 39 125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.6 30 67 38 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105 105 18 36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 61 32 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81: Duration of Action In Vivo of Oligonucleotides Targeting A1AT Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 72 were tested in a single dose study for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline and at 5, 12, 19, and 25 days following the dose. Plasma A1AT protein levels were measured via ELISA (see Example 80). The results below are presented as the average percent of plasma A1AT protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent lacking a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382, 678383, and 678384) were generally even more potent with even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656326).

TABLE 75 Plasma A1AT protein levels in mice Time point ISIS Dosage (days A1AT GalNAc₃ No. (mg/kg) post-dose) (% baseline) Cluster CM PBS n/a 5 93 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 62 25 77 656326 18 5 33 GalNAc₃-1a A_(d) 12 36 19 51 25 72 678381 18 5 21 GalNAc₃-3a A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a A_(d) 12 21 19 39 25 60 678383 18 5 24 GalNAc₃-10a A_(d) 12 21 19 45 25 73 678384 18 5 29 GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250 nM in Williams E medium and cells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16 hours following oligonucleotide addition, and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀ values were determined using Prism 4 software (GraphPad). The results show that oligonucleotides comprising a variety of different GalNAc conjugate groups and a variety of different cleavable moieties are significantly more potent in an in vitro free uptake experiment than the parent oligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and 666841).

TABLE 76 Inhibition of SRB-1 expression in vitro SEQ GalNAc IC₅₀ ID ISIS No. Sequence (5′ to 3′) Linkages cluster CM (nM) No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) PS n/a n/a 250 829 A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) PS GalNAc₃- A_(d) 40 830 A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) - 1_(a) GalNAc ₃-1 _(a) 661161 GalNAc ₃ -3 _(a) - _(o′) PS GalNAc₃- A_(d) 40 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 3_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 661162 GalNAc ₃ -3 _(a)-_(o′) PO/PS GalNAc₃- A_(d) 8 831 A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 3_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) PS GalNAc₃- A_(d) 20 830 A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) - 9_(a) GalNAc ₃ -9 _(a) 665001 GalNAc ₃ -8 _(a) - _(o′) PS GalNAc₃- A_(d) 70 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)- 8_(a) A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666224 GalNAc ₃ -5 _(a) - _(o′) PS GalNAc₃- A_(d) 80 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666841 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) PO/PS n/a n/a >250 829 A_(ds) ^(m)C_(ds)T_(ds )T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 666881 GalNAc ₃ -10 _(a-o′) PS GalNAc₃- A_(d) 30 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 10_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a-o′) PS GalNAc₃- PO 9 829 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) 3_(a) A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666924 GalNAc ₃ -3 _(a-o′) PS GalNAc₃- T_(d) 15 834 T _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 3_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666961 GalNAc ₃ -6 _(a-o′) PS GalNAc₃- A_(d) 150 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 6_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666981 GalNAc ₃ -7 _(a-o′) PS GalNAc₃- A_(d) 20 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 7_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670061 GalNAc ₃ -13 _(a-o′) PS GalNAc₃- A_(d) 30 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 13_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc ₃ -3 _(a-o′) PO/PS GalNAc₃- T_(d) 15 834 T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 3_(a) ^(m)C_(ds)A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 670700 GalNAc ₃ -3 _(a-o′) PO/PS GalNAc₃- A_(e) 30 831 A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 3_(a) ^(m)C_(ds)A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T 670701 GalNAc ₃ -3 _(a-o′) PO/PS GalNAc₃- T_(e) 25 834 T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 3_(a) ^(m)C_(ds)A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 671144 GalNAc ₃ -12 _(a-o′) PS GalNAc₃- A_(d) 40 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 12_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671165 GalNAc ₃ -13 _(a-o′) PO/PS GalNAc₃- A_(d) 8 831 A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 13_(a) ^(m)C_(ds)A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T 671261 GalNAc ₃ -14 _(a-o′) PS GalNAc₃- A_(d) >250 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 14_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671262 GalNAc ₃ -15 _(a-o′) PS GalNAc₃- A_(d) >250 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 15_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 673501 GalNAc ₃ -7 _(a-o′) PO/PS GalNAc₃- A_(d) 30 831 A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 7_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 673502 GalNAc ₃ -10 _(a-o′) PO/PS GalNAc₃- A_(d) 8 831 A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) 10_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 675441 GalNAc ₃ -17 _(a-o′) PS GalNAc₃- A_(d) 30 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 17_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675442 GalNAc ₃ -18 _(a-o′) PS GalNAc₃- A_(d) 20 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 18_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) PS GalNAc₃- A_(d) 40 830 A_(ds ) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) - 19_(a) GalNAc ₃-19 _(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) PS GalNAc₃- A_(d) 30 830 A_(ds ) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) - 20_(a) GalNAc ₃-20 _(a) 677843 GalNAc ₃ -23 _(a-o′) PS GalNAc₃- A_(d) 40 831 A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 23_(a) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds )T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown in Example 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-8_(a) was shown in Example 47, GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) was shown in Example 62, GalNAc₃-14_(a) was shown in Example 63, GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown in Example 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) was shown in Example 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a) was shown in Example 76.

Example 83: Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor XI Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor XI in mice.

TABLE 77 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQ No. Sequence (5′ to 3′) cluster CM ID No. 404071 T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 832 A_(es)G_(es)G_(e) 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo) GalNAc₃-1_(a) A_(d) 833 G_(eo)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663086 GalNAc ₃ -3 _(a) - _(o′) GalNAc₃-3_(a) A_(d) 841 A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃-7 _(a)-_(o′) GalNAc₃-7_(a) A_(d) 841 A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃ -10 _(a) - _(o′) GalNAc₃- A_(d) 841 A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) 10_(a) T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃ -13 _(a) - _(o′) GalNAc₃- A_(d) 841 A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) 13_(a) T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six to eight week old mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final dose. Factor XI liver mRNA levels were measured using real-time PCR and normalized to cyclophilin according to standard protocols. Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each treatment group, normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotides lowered Factor XI liver mRNA in a dose-dependent manner. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubin levels Factor XI Dosage mRNA (% ALT AST BUN Bilirubin GalNAc₃ SEQ ISIS No. (mg/kg) PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a 100 63 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 832 10 33 49 53 23 0.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 833 2 9 36 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16 GalNAc₃-3a 841 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 20 0.14 GalNAc₃-7a 841 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.7 39 43 54 21 0.16 GalNAc₃- 841 2 5 38 55 22 0.17 10a 6 2 25 38 20 0.14 678349 0.7 34 39 46 20 0.16 GalNAc₃- 841 2 8 43 63 21 0.14 13a 6 2 28 41 20 0.14

Example 84: Duration of Action In Vivo of Oligonucleotides Targeting Factor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.

Treatment

Six to eight week old mice were each injected subcutaneously once with an oligonucleotide listed in Table 77 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to determine baseline and at 3, 10, and 17 days following the dose. Plasma Factor XI protein levels were measured by ELISA using Factor XI capture and biotinylated detection antibodies from R & D Systems, Minneapolis, Minn. (catalog # AF2460 and # BAF2460, respectively) and the OptEIA Reagent Set B (Catalog #550534, BD Biosciences, San Jose, Calif.). The results below are presented as the average percent of plasma Factor XI protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79 Plasma Factor XI protein levels in mice Factor XI SEQ ISIS Dosage Time point (% GalNAc₃ ID No. (mg/kg) (days post-dose) baseline) Cluster CM No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/a n/a 832 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 833 10 3 17 21 663086 6 3 1 GalNAc₃-3a A_(d) 841 10 2 17 9 678347 6 3 1 GalNAc₃-7a A_(d) 841 10 1 17 8 678348 6 3 1 GalNAc₃-10a A_(d) 841 10 1 17 6 678349 6 3 1 GalNAc₃-13a A_(d) 841 10 1 17 5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 76 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of liver SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80 SRB-1 mRNA in liver ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94 GalNAc₃-1a A_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a A_(d) 0.3 107 1 68 3 26 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 666981 0.1 120 GalNAc₃-7a A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13a A_(d) 0.3 89 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 81 SRB-1 mRNA in liver ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM 661161 0.1 107 GalNAc₃-3a A_(d) 0.3 95 1 53 3 18 677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights were also measured using standard protocols. Average values for each treatment group are shown in Table 82 below.

TABLE 82 Body ISIS Dosage ALT AST Bilirubin BUN Weight GalNAc₃ No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster CM Saline n/a 19 39 0.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d) 0.3 29 56 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.1 35 83 0.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22 116 3 29 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d) 0.3 49 82 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.1 21 41 0.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22 111 3 77 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d) 0.3 20 57 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.1 20 38 0.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.15 21 119 3 41 57 0.14 23 118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in the tables below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Tail bleeds were performed at various time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 85-87. After the animals were sacrificed, plasma ALT, AST, and human TTR levels were measured, as were body weights, organ weights, and liver human TTR mRNA levels. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Tables 84-87 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. Body weights are the average percent weight change from baseline until sacrifice for each individual treatment group. Organ weights shown are normalized to the animal's body weight, and the average normalized organ weight for each treatment group is then presented relative to the average normalized organ weight for the PBS group.

In Tables 84-87, “BL” indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Tables 84 and 85, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the oligonucleotide comprising a GalNAc conjugate and full PS linkages.

TABLE 83 Oligonucleotides targeting human TTR SEQ GalNAc ID Isis No. Sequence 5′ to 3′ Linkages cluster CM No. 420915 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 842 A_(ds)A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃-1a A_(d) 843 A_(ds)A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -1 _(a) 682883 GalNAc ₃ -3 _(a-o′) PS/PO GalNAc₃-3a PO 842 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682884 GalNAc ₃ -7 _(a-o′) PS/PO GalNAc₃-7a PO 842 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682885 GalNAc ₃ -10 _(a-o′) PS/PO GalNAc₃- PO 842 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) 10a A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682886 GalNAc ₃ -13 _(a-o′) PS/PO GalNAc₃- PO 842 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) 13a A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684057 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS/PO GalNAc₃- A_(d) 843 A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19 _(a) 19a

The legend for Table 85 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62. The structure of GalNAc₃-19_(a) was shown in Example 70.

TABLE 84 Antisense inhibition of human TTR in vivo Plasma TTR TTR SEQ Isis Dosage mRNA protein GalNAc ID No. (mg/kg) (% PBS) (% PBS) cluster CM No. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 842 20 48 65 60 18 28 660261 0.6 113 87 GalNAc₃-1a A_(d) 843 2 40 56 6 20 27 20 9 11

TABLE 85 Antisense inhibition of human TTR in vivo Plasma TTR protein (% PBS at BL) TTR Day 17 SEQ Isis Dosage mRNA (After GalNAc ID No. (mg/kg) (% PBS) BL Day 3 Day 10 sac) cluster CM No. PBS n/a 100 100 96 90 114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 842 20 43 102 66 61 58 60 24 92 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃-3a PO 842 2 18 75 38 23 23 6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃-7a PO 842 2 19 76 44 25 23 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃-10a PO 842 2 22 93 58 32 32 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃-13a PO 842 2 21 89 50 31 30 6 18 102 41 24 27 684057 0.6 53 80 69 56 62 GalNAc₃-19a A_(d) 843 2 21 92 55 34 30 6 11 82 50 18 13

TABLE 86 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) SEQ Dosage Day Day Day Day Day Day Body Liver Spleen Kidney ID Isis No. (mg/kg) BL 3 10 17 BL 3 10 17 (% BL) (% PBS) (% PBS) (% PBS) No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a 420915 6 34 33 27 21 64 59 73 47 115 99 89 91 842 20 34 30 28 19 64 54 56 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 660261 0.6 33 38 28 26 70 71 63 59 111 96 99 92 843 2 29 32 31 34 61 60 68 61 118 100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 64 54 68 95 114 101 106 92

TABLE 87 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) SEQ Dosage Day Day Day Day Day Day Body Liver Spleen Kidney ID Isis No. (mg/kg) BL 3 10 17 BL 3 10 17 (% BL) (% PBS) (% PBS) (% PBS) No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a 420915 6 32 30 34 34 61 71 72 66 102 103 102 105 842 20 41 34 37 33 80 76 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99 682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 842 2 38 39 42 43 71 84 70 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 682884 0.6 33 32 35 34 70 74 75 67 101 100 130 99 842 2 31 32 38 38 63 77 66 55 104 103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 26 37 35 63 63 77 59 100 109 109 112 842 2 30 26 38 40 54 56 71 72 102 98 111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34 36 58 87 54 61 104 99 120 101 842 2 27 26 34 36 51 55 55 69 103 91 105 92 6 40 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 56 51 51 69 104 99 110 102 843 2 33 32 31 40 54 57 56 87 103 100 112 97 6 39 33 35 40 67 52 55 92 98 104 121 108

Example 87: Duration of Action In Vivo by Single Doses of Oligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS numbers 420915, 682883, and 682885 (see Table 83) were also tested in a single dose study for duration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 88 Plasma TTR protein levels Time point SEQ ISIS Dosage (days post- TTR GalNAc₃ ID No. (mg/kg) dose) (% baseline) Cluster CM No. 420915 100 3 30 n/a n/a 842 7 23 10 35 17 53 24 75 39 100 660261 13.5 3 27 GalNAc₃-1a A_(d) 843 7 21 10 22 17 36 24 48 39 69 Treatment

Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 89 Plasma TTR protein levels Time point SEQ ISIS Dosage (days post- TTR GalNAc₃ ID No. (mg/kg) dose) (% baseline) Cluster CM No. 420915 100 3 48 n/a n/a 842 7 48 10 48 17 66 31 80 682883 10.0 3 45 GalNAc₃-3a PO 842 7 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO 842 7 33 10 34 17 40 31 64

The results in Tables 88 and 89 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer duration of action than the parent oligonucleotide lacking a conjugate (ISIS 420915).

Example 88: Splicing Modulation In Vivo by Oligonucleotides Targeting SMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.

TABLE 90 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es) n/a n/a 844 G_(e) 699819 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es) GalNAc₃- PO 844 T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)G_(e) 7a 699821 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(eo) ^(m)C_(eo)T_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)T_(eo)A_(eo) GalNAc₃- PO 844 A_(eo)T_(eo)G_(eo) ^(m)C_(eo)T_(es)G_(es)G_(e) 7a 700000 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es) GalNAc₃- A_(d) 845 G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 1a 703421 X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 844 703422 GalNAc ₃ -7 _(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃- n/a 844 7b

The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X” indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.), and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the —NH—C₆—O portion of the linker as shown below:

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide.

Treatment

Six week old transgenic mice that express human SMN were injected subcutaneously once with an oligonucleotide listed in Table 91 or with saline. Each treatment group consisted of 2 males and 2 females. The mice were sacrificed 3 days following the dose to determine the liver human SMN mRNA levels both with and without exon 7 using real-time PCR according to standard protocols. Total RNA was measured using Ribogreen reagent. The SMN mRNA levels were normalized to total mRNA, and further normalized to the averages for the saline treatment group. The resulting average ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table 91. The results show that fully modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in altering splicing in the liver than the parent oligonucleotides lacking a GlaNAc conjugate. Furthermore, this trend is maintained for multiple modification chemistries, including 2′-MOE and morpholino modified oligonucleotides.

TABLE 91 Effect of oligonucleotides targeting human SMN in vivo ISIS Dose +Exon GalNAc₃ SEQ ID No. (mg/kg) 7/−Exon 7 Cluster CM No. Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 844 387954 288 5.00 n/a n/a 844 699819 32 7.84 GalNAc₃-7a PO 844 699821 32 7.22 GalNAc₃-7a PO 844 700000 32 6.91 GalNAc₃-1a A_(d) 845 703421 32 1.27 n/a n/a 844 703422 32 4.12 GalNAc₃-7b n/a 844

Example 89: Antisense Inhibition In Vivo by Oligonucleotides Targeting Apolipoprotein a (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m) n/a n/a 847 C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds) G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es) T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m) GalNAc₃-7a PO 847 C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds) T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once per week at a dosage shown below, for a total of six doses, with an oligonucleotide listed in Table 92 or with PBS. Each treatment group consisted of 3-4 animals. Tail bleeds were performed the day before the first dose and weekly following each dose to determine plasma Apo(a) protein levels. The mice were sacrificed two days following the final administration. Apo(a) liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma protein results in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were further normalized to the baseline (BL) value for the PBS group. Average absolute transaminase levels and body weights (% relative to baseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the oligonucleotides lowered Apo(a) liver mRNA and plasma protein levels in a dose-dependent manner. Furthermore, the oligonucleotide comprising the GalNAc conjugate was significantly more potent with a longer duration of action than the parent oligonucleotide lacking a GalNAc conjugate. As illustrated in Table 94, transaminase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.

TABLE 93 Apo(a) liver mRNA and plasma protein levels Apo(a) Dosage mRNA Apo(a) plasma protein (% PBS) ISIS No. (mg/kg) (% PBS) BL Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 PBS n/a 100 100 120 119 113 88 121 97 494372 3 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 7 9 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 82 52 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 94 ISIS Dosage ALT AST Body weight No. (mg/kg) (U/L) (U/L) (% baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103 681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in Table 96 or with PBS. Each treatment group consisted of 4 animals. Prior to the first dose, a tail bleed was performed to determine plasma TTR protein levels at baseline (BL). The mice were sacrificed 72 hours following the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Table 96 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. “BL” indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Table 96, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or deoxyadenosine cleavable moiety showed significant improvements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95 Oligonucleotides targeting human TTR SEQ GalNAc ID Isis No. Sequence 5′ to 3′ Linkages cluster CM No. 420915 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 842 A_(ds)A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′) PS/PO GalNAc₃-3a PO 842 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′) PS/PO GalNAc₃-3a A_(d) 846 A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682887 GalNAc ₃ -7 _(a-o′) PS/PO GalNAc₃-7a A_(d) 846 A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(as)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) PS/PO GalNAc₃- A_(d) 846 A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) 10a ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) PS/PO GalNAc₃- A_(d) 846 A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) 13a ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e)

The legend for Table 95 can be found in Example 74. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 96 Antisense inhibition of human TTR in vivo Isis Dosage TTR mRNA TTR protein GalNAc No. (mg/kg) (% PBS) (% BL) cluster CM PBS n/a 100 124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73 GalNAc₃-3a PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a A_(d) 2 33 57 6 17 22 682887 0.6 60 97 GalNAc₃-7a A_(d) 2 36 49 6 12 19 682888 0.6 65 92 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13a A_(d) 2 38 45 6 16 18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 97 below were tested in a non-terminal, dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 97 or with PBS. Each treatment group consisted of 4 males and 1 female. Prior to the first dose and at various time points thereafter, blood draws were performed to determine plasma Factor VII protein levels. Factor VII protein levels were measured by ELISA. The results presented in Table 98 are the average values for each treatment group relative to the average value for the PBS group at baseline (BL), the measurements taken just prior to the first dose. As illustrated in Table 98, treatment with antisense oligonucleotides lowered Factor VII expression levels in a dose-dependent manner, and the oligonucleotide comprising the GalNAc conjugate was significantly more potent in monkeys compared to the oligonucleotide lacking a GalNAc conjugate.

TABLE 97 Oligonucleotides targeting Factor VII SEQ Isis GalNAc ID No. Sequence 5′ to 3′ Linkages cluster CM No. 407935 A_(es)T_(es)G_(es) ^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)T_(ds)G_(ds) ^(m)C_(ds) PS n/a n/a 848 T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e) 686892 GalNAc ₃ -10 _(a-o′) PS GalNAc₃- PO 848 A_(es)T_(es)G_(es) ^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) 10a A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)

The legend for Table 97 can be found in Example 74. The structure of GalNAc₃-10_(a) was shown in Example 46.

TABLE 98 Factor VII plasma protein levels ISIS Dose Factor VII No. Day (mg/kg) (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 46 43 n/a 43 686892 0  3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a 11

Example 92: Antisense Inhibition in Primary Hepatocytes by Antisense Oligonucleotides Targeting Apo-CIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 99, targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or 333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24 hours, the cells were lysed and total RNA was purified using RNeasy (Qiagen). ApoC-III mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) according to standard protocols. IC₅₀ values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a phosphodiester-linked deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide lacking a conjugate.

TABLE 99 Inhibition of mouse APOC-III expression in mouse primary hepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m) n/a 13.20 849 C_(es)A_(e) 661180 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(d) 1.40 850 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) PO 0.70 849 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7 _(a-o′) PO 1.70 849 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10 _(a-o′) PO 2.00 849 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13 _(a-o′) PO 1.50 849 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3 _(a-o′) PO <0.46 849 ^(m)C_(es)A_(eo)G_(eo) ^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(eo) A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3 _(a) - A_(d) 1.10 851 _(o′) A _(do) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(d) 2.50 850 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-13_(a) was shown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 100 Modified ASOs targeting SRB-1 GalNAc₃ SEQ Isis No. Sequence (5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds )A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 852 699806 GalNAc ₃ -3 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃- PO 852 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 3a 699807 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃- PO 852 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 7a 699809 GalNAc ₃ -7 _(a) - _(o′) T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds )A_(ds) ^(m)C_(ds) GalNAc₃- PO 852 T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 7a 699811 GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃- PO 852 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 7a 699813 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ds) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃- PO 852 T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 7a 699815 GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds )A_(ds)T_(ds )G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃- PO 852 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e) 7a

The structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48. Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO). Supersript “m” indicates 5-methylcytosines.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 100 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-1 mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 101, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the gapmer oligonucleotides comprising a GalNAc conjugate and having wings that were either full cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were also measured, and the average values for each treatment group are shown in Table 101. Body weight is shown as the average percent body weight relative to the baseline body weight (% BL) measured just prior to the oligonucleotide dose.

TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights SRB-1 Body ISIS Dosage mRNA ALT AST weight No. (mg/kg) (% PBS) (U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 18 48 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1 114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104 699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 69 0.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 1 33 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.13 25 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 22 61 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 105 0.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 102 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequence (5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(esAds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 829 T_(es)T_(e) 700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms) n/a n/a 853 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃- PO 829 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3a 700991 GalNAc ₃ -7 _(a) - _(o)′G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃- PO 853 A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m) 7a

Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example 74 for complete table legend. The structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 103 below and show that both the 2′-MOE and 2′-OMe modified oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides lacking a conjugate. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 103 SRB-1 mRNA ISIS Dosage SRB-1 mRNA No. (mg/kg) (% PBS) PBS n/a 100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 45 10 17 700991 1 118 3 63 10 14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 104 Modified ASOs targeting SRB-1 SEQ ISIS GalNAc₃ ID No. Sequences (5′ to 3′) Cluster CM No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a n/a 823 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 823 699782 GalNAc ₃ -7 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-7_(a) PO 823 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls) ^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 823 653621 T_(ls) ^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′) -GalNAc ₃ -1 _(a) GalNAc₃-1_(a) A_(d) 824 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d )G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/a n/a 823 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d )G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 823

Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicates a locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74 table legend for other abbreviations. The structure of GalNAc₃-1_(a) was shown previously in Example 9, the structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 105 below and show that oligonucleotides comprising a GalNAc conjugate and various bicyclic nucleoside modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising bicyclic nucleoside modifications. Furthermore, the oligonucleotide comprising a GalNAc conjugate and fluoro-HNA modifications was significantly more potent than the parent lacking a conjugate and comprising fluoro-HNA modifications. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 440762 1 104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15 699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 77 10 37 699789 0.1 82 0.3 69 1 26

Example 96: Plasma Protein Binding of Antisense Oligonucleotides Comprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.

TABLE 106 Modified oligonucleotides targeting Apo(a) SEQ ISIS GalNAc₃ ID No. Sequences (5′ to 3′) Cluster CM No 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 847 T_(es) ^(m)C_(e) 693401 T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 847 T_(es) ^(m)C_(e) 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) GalNAc₃- PO 847 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 7a 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) GalNAc₃- PO 847 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 7a See the Example 74 for table legend. The structure of GalNAc₃-7a was shown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regenerated cellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with 300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, then with 300 μL of a 300 μg/mL solution of a control oligonucleotide in H₂O and centrifuged at 2000 g for 16 minutes. In order to assess non-specific binding to the filters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mL solution of oligonucleotide in H₂O at pH 7.4 was placed in the pre-conditioned filters and centrifuged at 2000 g for 16 minutes. The unfiltered and filtered samples were analyzed by an ELISA assay to determine the oligonucleotide concentrations. Three replicates were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample is used to determine the percent of oligonucleotide that is recovered through the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-free human volunteers, cynomolgus monkeys, and CD-1 mice, were purchased from Bioreclamation LLC (Westbury, N.Y.). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37° C. for 30 minutes, immediately followed by centrifugation at 2000 g for 16 minutes. Aliquots of filtered and unfiltered spiked plasma samples were analyzed by an ELISA to determine the oligonucleotide concentration in each sample. Three replicates per concentration were used to determine the average percentage of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample is used to determine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (% unbound). The final unbound oligonucleotide values are corrected for non-specific binding by dividing the % unbound by the % recovery for each oligonucleotide. The final % bound oligonucleotide values are determined by subtracting the final % unbound values from 100. The results are shown in Table 107 for the two concentrations of oligonucleotide tested (5 and 150 μg/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a significant impact on plasma protein binding. Furthermore, oligonucleotides with full PS internucleoside linkages and mixed PO/PS linkages both bind plasma proteins, and those with full PS linkages bind plasma proteins to a somewhat greater extent than those with mixed PO/PS linkages.

TABLE 107 Percent of modified oligonucleotide bound to plasma proteins Human plasma Monkey plasma Mouse plasma ISIS No. 5 μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2 663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.6 89.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.0 94.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.6 93.7 95.6 92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃ Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were designed to target TTR.

TABLE 108 Modified oligonucleotides targeting TTR ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a-o′) A _(do )T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds) GalNAc₃-3 A_(d) 846 ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 666942 T_(es ) ^(m)C_(eo )T_(eo )T_(eo )G_(eo )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds) GalNAc₃-1 A_(d) 843 A_(ds )A_(ds )A_(eo )T_(eo ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(eo ) A _(do′) -GalNAc ₃ -3 _(a) 682876 GalNAc ₃ -3 _(a-o′)T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds) GalNAc₃-3 PO 842 A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682877 GalNAc ₃ -7 _(a-o′)T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds) GalNAc₃-7 PO 842 A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682878 GalNAc ₃ -10 _(a-o′)T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds) GalNAc₃-10 PO 842 A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682879 GalNAc ₃ -13 _(a-o′)T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds) GalNAc₃-13 PO 842 A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682880 GalNAc ₃ -7 _(a-o′) A _(do )T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds) GalNAc₃-7 A_(d) 846 ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682881 GalNAc ₃ -10 _(a-o′) A _(do )T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds) GalNAc₃-10 A_(d) 846 ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682882 GalNAc ₃ -13 _(a-o′) A _(do )T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds) GalNAc₃-13 A_(d) 846 ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 684056 T_(es ) ^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds) GalNAc₃-19 A_(d) 846 A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(eo ) A _(do′) -GalNAc ₃ -19 _(a)

The legend for Table 108 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62. The structure of GalNAc₃-19_(a) was shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of Oligonucleotides Comprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested for pro-inflammatory effects in an hPMBC assay as described in Examples 23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the oligonucleotides.) ISIS 353512 is a high responder used as a positive control, and the other oligonucleotides are described in Tables 83, 95, and 108. The results shown in Table 109 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS internucleoside linkages produced significantly lower pro-inflammatory responses compared to the same oligonucleotides having full PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.

TABLE 109 ISIS No. E_(max)/EC₅₀ GalNAc₃ cluster Linkages CM 353512 3630 n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 682888 0.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAc Conjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 110 (see Table 76 for descriptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competitor ligand, al-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 37° C., and >90% desialylation was confirmed by either sialic acid assay or size exclusion chromatography (SEC). Iodine monochloride was used to iodinate the AGP according to the procedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1):173-81.) In this method, desialylated al-acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na¹²⁵I, and 1 M glycine in 0.25 M NaOH. After incubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP was separated from free ¹²⁵I by concentrating the mixture twice utilizing a 3 KDMWCO spin column. The protein was tested for labeling efficiency and purity on a HPLC system equipped with an Agilent SEC-3 column (7.8×300 mm) and a 13-RAM counter. Competition experiments utilizing ¹²⁵I-labeled de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (10⁶ cells/ml) were plated on 6-well plates in 2 ml of appropriate growth media. MEM media supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used. Cells were incubated 16-20 hours @ 37° C. with 5% and 10% CO₂ respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37° C. with 1 ml competition mix containing appropriate growth media with 2% FBS, 10⁻⁸ M ¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10⁻¹¹ to 10⁻⁵ M. Non-specific binding was determined in the presence of 10⁻² M GalNAc sugar. Cells were washed twice with media without FBS to remove unbound ¹²⁵I-labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen's RLT buffer containing 1% ß-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a γ-counter. Non-specific binding was subtracted before dividing ¹²⁵I protein counts by the value of the lowest GalNAc-ASO concentration counts. The inhibition curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to calculate the binding affinities (K_(D)'s).

The results in Table 110 were obtained from experiments performed on five different days. Results for oligonucleotides marked with superscript “a” are the average of experiments run on two different days. The results show that the oligonucleotides comprising a GalNAc conjugate group on the 5′-end bound the asialoglycoprotein receptor on human HepG2 cells with 1.5 to 16-fold greater affinity than the oligonucleotides comprising a GalNAc conjugate group on the 3′-end.

TABLE 110 Asialoglycoprotein receptor binding assay results Oligonucleotide end to which GalNAc ISIS No. GalNAc conjugate conjugate is attached K_(D) (nM) 661161^(a) GalNAc₃-3 5′ 3.7 666881^(a) GalNAc₃-10 5′ 7.6 666981 GalNAc₃-7 5′ 6.0 670061 GalNAc₃-13 5′ 7.4 655861^(a) GalNAc₃-1 3′ 11.6 677841^(a) GalNAc₃-19 3′ 60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.

TABLE 111a Modified ASOs targeting APO(a) GalNAc₃ SEQ ISIS No. Sequences (5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Female transgenic mice that express human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an oligonucleotide and dosage listed in Table 111b or with PBS. Each treatment group consisted of 3 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 72 hours, 1 week, and 2 weeks following the first dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the first dose. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 111b are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the oligonucleotides comprising a GalNAc conjugate group exhibited potent reduction in Apo(a) expression. This potent effect was observed for the oligonucleotide that comprises full PS internucleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.

TABLE 111b Apo(a) plasma protein levels Apo(a) ISIS Dosage at 72 hours Apo(a) at 1 week Apo(a) at 3 weeks No. (mg/kg) (% BL) (% BL) (% BL) PBS n/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 23 15 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101: Antisense Inhibition by Oligonucleotides Comprising a GalNAc Cluster Linked Via a Stable Moiety

The oligonucleotides listed in Table 112 were tested for inhibition of mouse APOC-III expression in vivo. C57Bl/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 112 or with PBS. Each treatment group consisted of 4 animals. Each mouse treated with ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mouse treated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847 is linked via a stable moiety, a phosphorothioate linkage instead of a readily cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using real-time PCR. APOC-III mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented in Table 112 as the average percent of APOC-III mRNA levels for each treatment group relative to the saline control group. The results show that the oligonucleotides comprising a GalNAc conjugate group were significantly more potent than the oligonucleotide lacking a conjugate group. Furthermore, the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a cleavable moiety (ISIS 680772) was even more potent than the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 112 Modified oligonucleotides targeting mouse APOC-III APOC-III SEQ ISIS Dosage mRNA (% ID No. Sequences (5′ to 3′) CM (mg/kg) PBS) No. 440670 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 849 G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 20 59 60 37 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds) PO 0.6 79 849 T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es )A_(es)G_(es) ^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃ -7 _(a-s′) n/a 0.6 83 849 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds) (PS) 2 73 T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es )A_(es)G_(es) ^(m)C_(es)A_(e) 6 40 20 28

The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102: Distribution in Liver of Antisense Oligonucleotides Comprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 36) that does not comprise a GalNAc conjugate and ISIS 655861 (see Table 36) that does comprise a GalNAc conjugate was evaluated. Male balb/c mice were subcutaneously injected once with ISIS 353382 or 655861 at a dosage listed in Table 113. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655861, which consisted of 2 animals. The animals were sacrificed 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide molecules per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide probe used to detect the antisense oligonucleotides. The results presented in Table 113 are the average concentrations of oligonucleotide for each treatment group in units of millions of oligonucleotide molecules per cell. The results show that at equivalent doses, the oligonucleotide comprising a GalNAc conjugate was present at higher concentrations in the total liver and in hepatocytes than the oligonucleotide that does not comprise a GalNAc conjugate. Furthermore, the oligonucleotide comprising a GalNAc conjugate was present at lower concentrations in non-parenchymal liver cells than the oligonucleotide that does not comprise a GalNAc conjugate. And while the concentrations of ISIS 655861 in hepatocytes and non-parenchymal liver cells were similar per cell, the liver is approximately 80% hepatocytes by volume. Thus, the majority of the ISIS 655861 oligonucleotide that was present in the liver was found in hepatocytes, whereas the majority of the ISIS 353382 oligonucleotide that was present in the liver was found in non-parenchymal liver cells.

TABLE 113 Concentration Concentration in in whole Concentration in non-parenchymal liver hepatocytes liver cells (molecules* (molecules* (molecules* ISIS Dosage 10{circumflex over ( )}6 10{circumflex over ( )}6 per 10{circumflex over ( )}6 per No. (mg/kg) per cell) cell) cell) 353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1

Example 103: Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 114 below were tested in a single dose study for duration of action in mice.

TABLE 114 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 821 T_(es)A_(es)T_(e) 663084 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 837 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo )T_(es)A_(es)T_(e) 679241 A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 822 T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The structure of GalNAc₃-3_(a) was shown in Example 39, and GalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 114 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 115 are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels. A comparison of the results in Table 71 of example 79 with the results in Table 115 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate internucleoside linkages exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.

TABLE 115 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point (days APOC-III ISIS Dosage post- Triglycerides protein GalNAc₃ No. (mg/kg) dose) (% baseline) (% baseline) Cluster CM PBS n/a 3 96 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 72 88 304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 42 82 92 663084 10 3 35 28 GalNAc₃-3a A_(d) 7 23 24 14 23 26 21 23 29 28 30 22 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃-19a A_(d) 7 31 28 14 30 22 21 36 34 28 48 34 35 50 45 42 72 64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc₂ Conjugate

Compound 120 is commercially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF (10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction. After 3 h, the reaction mixture was poured into 100 mL of 1 M NaHSO4 and extracted with 2×50 mL ethyl acetate. Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and 2× brine, dried with Na₂SO₄, filtered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) to yield compound 231. LCMS and NMR were consistent with the structure. Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 mL) and trifluoracetic acid (10 mL) was added. After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3×10 mL). The residue was dried under reduced pressure to yield compound 232 as the trifuloracetate salt. The synthesis of compound 166 is described in Example 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and N,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirred at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was extracted with EtOAc (2×100 mL). The organic phase was separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 M NaHSO₄ (3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield compound 233. LCMS and NMR were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt % Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The reaction mixture was filtered through a pad of Celite and concentrated to yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolved in DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring at room temperature the reaction mixture was poured into water (40 mL) and extracted with EtOAc (2×50 mL). A standard work-up was completed as described above to yield compound 234. LCMS and NMR were consistent with the structure. Oligonucleotide 235 was prepared using the general procedure described in Example 46. The GalNAc₂ cluster portion (GalNAc₂-24_(a)) of the conjugate group GalNAc₂-24 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₂-24 (GalNAc₂-24_(a)-CM) is shown below:

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc₁-25 Conjugate

The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared using the general procedure described in Example 46.

Alternatively, oligonucleotide 236 was synthesized using the scheme shown below, and compound 238 was used to form the oligonucleotide 236 using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate group GalNAc₁-25 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₂ or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested in dose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222, or 708561; or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the ED₅₀ results are presented in Tables 116 and 117. Although previous studies showed that trivalent GalNAc-conjugated oligonucleotides were significantly more potent than divalent GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than monovalent GalNAc conjugated oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides comprising monovalent, divalent, and trivalent GalNAc clusters lowered SRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116 Modified oligonucleotides targeting SRB-1 SEQ ISIS GalNAc ED₅₀ ID No. Sequences (5′ to 3′) Cluster (mg/kg) No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 823 686221 GalNAc ₂ -24 _(a) - _(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 827 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃ -13 _(a) - _(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 827 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

See Example 93 for table legend. The structure of GalNAc₃-13a was shown in Example 62, and the structure of GalNAc₂-24a was shown in Example 104.

TABLE 117 Modified oligonucleotides targeting SRB-1 SEQ ISIS GalNAc ED₅₀ ID No. Sequences (5′ to 3′) Cluster (mg/kg) No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 823 708561 GalNAc ₁ -25 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 823 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

See Example 93 for table legend. The structure of GalNAc₁-25a was shown in Example 105.

The concentrations of the oligonucleotides in Tables 116 and 117 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 117a and 117b below are the average total antisense oligonucleotide tissues levels for each treatment group, as measured by UV in units of μg oligonucleotide per gram of liver tissue. The results show that the oligonucleotides comprising a GalNAc conjugate group accumulated in the liver at significantly higher levels than the same dose of the oligonucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc ligands in their respective conjugate groups all accumulated in the liver at similar levels. This result is surprising in view of the Khorev et al. literature reference cited above and is consistent with the activity data shown in Tables 116 and 117 above.

TABLE 117a Liver concentrations of oligonucleotides comprising a GalNAc₂ or GalNAc₃ conjugate group Dosage [Antisense ISIS No. (mg/kg) oligonucleotide] (μg/g) GalNAc cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1 686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5 GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 117b Liver concentrations of oligonucleotides comprising a GalNAc₁ conjugate group Dosage [Antisense ISIS No. (mg/kg) oligonucleotide] (μg/g) GalNAc cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7 708561 0.2 0.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc₁-26 or GalNAc₁-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. The resulting amide containing compound is phosphitylated, then added to the 5′-end of an oligonucleotide using procedures described in Example 10. The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate group GalNAc₁-26 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of an oligonucleotide, the amide formed from the reaction of compounds 47 and 64 is added to a solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate group GalNAc₁-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a single dose study in mice.

TABLE 118 Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) n/a n/a 847 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es )T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃ -3 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo )T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃ -10 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m )C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo )T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo )T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃ -13 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 847 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo )T_(es)T_(es) ^(m)C_(e) 681260 T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds )T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 854 T_(es)T_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 119 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 1 week following the first dose. Additional blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 119 are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides reduced Apo(a) protein expression. Furthermore, the oligonucleotides comprising a GalNAc conjugate group exhibited even more potent reduction in Apo(a) expression than the oligonucleotide that does not comprise a conjugate group.

TABLE 119 Apo(a) plasma protein levels ISIS Dosage Apo(a) at 1 week No. (mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 681256 10 17 681257 10 24 681258 10 22 681260 10 26

Example 109: Synthesis of Oligonucleotides Comprising a GalNAc₁-28 or GalNAc₁-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures described in Example 10 to synthesize the oligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of the conjugate group GalNAc₁-28 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-28 (GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of an oligonucleotide, procedures similar to those described in Example 71 are used to form the hydroxyl intermediate, which is then added to the solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate group GalNAc₁-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30 Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of the conjugate group GalNAc₁-30 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, Y is part of the cleavable moiety. In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₁-30_(a) is shown below:

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc₂-31 or GalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂ cluster portion (GalNAc₂-31,) of the conjugate group GalNAc₂-31 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₂-31_(a) is shown below:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate is shown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of the conjugate group GalNAc₂-32 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₂-32_(a) is shown below:

Example 112: Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized with a GalNAc₁ conjugate group in order to further test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

TABLE 120 SEQ ISIS GalNAc ID No. Sequence (5′ to 3′) cluster CM NO. 711461 GalNAc ₁ -25 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds) GalNAc₁- A_(d) 831 A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 25_(a) 711462 GalNAc ₁ -25 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁- PO 829 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 25_(a) 711463 GalNAc ₁ -25 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁- PO 829 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 25_(a) 711465 GalNAc ₁ -26 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds) GalNAc₁- A_(d) 831 A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 26_(a) 711466 GalNAc ₁ -26 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁- PO 829 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 26_(a) 711467 GalNAc ₁ -26 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁- PO 829 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 26_(a) 711468 GalNAc ₁ -28 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds) GalNAc₁- A_(d) 831 A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 28_(a) 711469 GalNAc ₁ -28 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁- PO 829 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 28_(a) 711470 GalNAc ₁ -28 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁- PO 829 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 28_(a) 713844 G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds) GalNAc₁- PO 829 T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -27 _(a) 27_(a) 713845 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds) GalNAc₁- PO 829 ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -27 _(a) 27_(a) 713846 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds) GalNAc₁- A_(d) 830 ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁ -27 _(a) 27a 713847 G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds) GalNAc₁- PO 829 T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -29 _(a) 29_(a) 713848 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds) GalNAc₁- PO 829 ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -29 _(a) 29_(a) 713849 G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds) GalNAc₁- A_(d) 830 T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁ -29 _(a) 29_(a) 713850 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds) GalNAc₁- A_(d) 830 ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁ -29 _(a) 29_(a)

Example 113: Antisense Inhibition In Vivo by Oligonucleotides Targeting CFB

The oligonucleotides listed in Table 121 were tested in a dose-dependent study for antisense inhibition of human Complement Factor B (CFB) in mice.

TABLE 121 Modified ASOs targeting CFB ISIS No. Sequences (5′ to 3′) GalNAc₃ Cluster CM SEQ ID No. 588540 A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(ds) ^(m)C_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) n/a n/a 440 ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(es) ^(m)C_(es)A_(es)G_(es) ^(m)C_(e) 687301 GalNAc ₃-3 _(a) - _(o′)A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(ds) ^(m)C_(ds)G_(ds) GalNAc₃-3a PO 440 ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(es) ^(m)C_(es)A_(es)G_(es) ^(m)C_(e)

The structure of GalNAc₃-3_(a) was shown previously in Example 39.

Treatment

Transgenic mice that express human CFB (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once per week for 3 weeks (a total of 4 doses) with an oligonucleotide listed in Table 122 or with saline. The four treatment groups that received ISIS No. 588540 were given 6, 12, 25, or 50 mg/kg per dose. The four treatment groups that received ISIS No. 687301 were given 0.25, 0.5, 2, or 6 mg/kg per dose. Each treatment group consisted of 4 animals. The mice were sacrificed 2 days following the final administration to determine the liver and kidney human CFB and cyclophilin mRNA levels using real-time PCR according to standard protocols. The CFB mRNA levels were normalized to the cyclophilin levels, and the averages for each treatment group were used to determine the dose that achieved 50% inhibition of the human CFB transcript expression (ED₅₀). The results are the averages of four experiments completed with two different primer probe sets and are shown in Table 122.

TABLE 122 Potencies of oligonucleotides targeting human CFB in vivo ED₅₀ in liver ED₅₀ in kidney GalNAc₃ ISIS No. (mg/kg) (mg/kg) Cluster CM 588540 7.9 11.7 n/a n/a 687301 0.49 0.35 GalNAc₃-3a PO

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin, BUN, and body weights were also evaluated. The results show that there were no significant changes in any of the treatment groups relative to the saline treated group (data not shown), indicating that both oligonucleotides were very well tolerated.

Example 114: Antisense Inhibition In Vivo by Oligonucleotides Targeting CFB

The oligonucleotides listed in Table 123 were tested in a dose-dependent study for antisense inhibition of human CFB in mice.

Treatment

Transgenic mice that express human CFB (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once with 0.6, 1, 6, or 18 mg/kg of an oligonucleotide listed in Table 123 or with saline. Each treatment group consisted of 4 or 5 animals. The mice were sacrificed 72 hours following the dose to determine the liver human CFB and cyclophilin mRNA levels using real-time PCR according to standard protocols. The CFB mRNA levels were normalized to the cyclophilin levels, and the averages for each treatment group were used to determine the dose that achieved 50% inhibition of the human CFB transcript expression (ED₅₀). The results are shown in Table 123.

TABLE 123 Modified ASOs targeting CFB ED₅₀ in ISIS GalNAc₃ liver SEQ No. Sequences (5′ to 3′) Cluster CM (mg/kg) ID No. 696844 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(ds) ^(m)C_(ds)G_(ds) GalNAc₃-7a PO 0.86 440 ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(es) ^(m)C_(es)A_(es)G_(es) ^(m)C_(e) 696845 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(eo) ^(m)C_(eo) ^(m)C_(eo) ^(m)C_(eo)A_(ds) ^(m)C_(ds)G_(ds) GalNAc₃-7a PO 0.71 440 ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(eo) ^(m)C_(eo)A_(es)G_(es) ^(m)C_(e) 698969 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(eo) ^(m)C_(eo) ^(m)C_(eo) ^(m)C_(es)A_(ds) ^(m)C_(ds)G_(ds) GalNAc₃-7a PO 0.51 440 ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(eo) ^(m)C_(eo)A_(es)G_(es) ^(m)C_(e) 698970 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(es) ^(m)C_(eo) ^(m)C_(eo) ^(m)C_(eo)A_(ds) ^(m)C_(ds)G_(ds) GalNAc₃-7a PO 0.55 440 ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(eo) ^(m)C_(eo)A_(es)G_(es) ^(m)C_(e)

The structure of GalNAc₃-7_(a) was shown previously in Example 48.

Example 115: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 (forward sequence AGTCTCTGTGGCATGGTTTGG, designated herein as SEQ ID NO: 810; reverse sequence GGGCGAATGACTGAGATCTTG, designated herein as SEQ ID NO: 811; probe sequence TACCGATTACCACAAGCAACCATGGCA, designated herein as SEQ ID NO: 812) was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 124 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site Region Sequence inhibition site site NO: 532608 20 39 Exon 1 GCTGAGCTGCCAGTCAAGGA 36 1741 1760 6 532609 26 45 Exon 1 GGCCCCGCTGAGCTGCCAGT 16 1747 1766 7 532610 45 64 Exon 1 CGGAACATCCAAGCGGGAGG 11 1766 1785 8 532611 51 70 Exon 1 CTTTCCCGGAACATCCAAGC 26 1772 1791 9 532612 100 119 Exon 1 ATCTGTGTTCTGGCACCTGC 25 1821 1840 10 532613 148 167 Exon 1 GTCACATTCCCTTCCCCTGC 39 1869 1888 11 532614 154 173 Exon 1 GACCTGGTCACATTCCCTTC 71 1875 1894 12 532615 160 179 Exon 1 GACCTAGACCTGGTCACATT 35 1881 1900 13 532616 166 185 Exon 1 ACTCCAGACCTAGACCTGGT 39 1887 1906 14 532617 172 191 Exon 1 GCTGAAACTCCAGACCTAGA 27 1893 1912 15 532618 178 197 Exon 1 GTCCAAGCTGAAACTCCAGA 29 1899 1918 16 532619 184 203 Exon 1 CTCAGTGTCCAAGCTGAAAC 21 1905 1924 17 532620 246 265 Exon 1 AGGAGAGAAGCTGGGCCTGG 31 1967 1986 18 532621 252 271 Exon 1 GAAGGCAGGAGAGAAGCTGG 25 1973 1992 19 532622 336 355 Exon 1-2 GTGGTGGTCACACCTCCAGA 28 n/a n/a 20 Junction 532623 381 400 Exon 2 CCCTCCAGAGAGCAGGATCC 22 2189 2208 21 532624 387 406 Exon 2 TCTACCCCCTCCAGAGAGCA 37 2195 2214 22 532625 393 412 Exon 2 TTGATCTCTACCCCCTCCAG 30 2201 2220 23 532626 417 436 Exon 2 TGGAGAAGTCGGAAGGAGCC 35 2225 2244 24 532627 423 442 Exon 2 CCCTCTTGGAGAAGTCGGAA 37 2231 2250 25 532628 429 448 Exon 2 GCCTGGCCCTCTTGGAGAAG 0 2237 2256 26 532629 435 454 Exon 2 TCCAGTGCCTGGCCCTCTTG 26 2243 2262 27 532630 458 477 Exon 2 AGAAGCCAGAAGGACACACG 30 2266 2285 28 532631 464 483 Exon 2 ACGGGTAGAAGCCAGAAGGA 43 2272 2291 29 532632 480 499 Exon 2 CGTGTCTGCACAGGGTACGG 57 2288 2307 30 532633 513 532 Exon 2 AGGGTGCTCCAGGACCCCGT 27 2321 2340 31 532634 560 579 Exon 2-3 TTGCTCTGCACTCTGCCTTC 41 n/a n/a 32 Junction 532635 600 619 Exon 3 TATTCCCCGTTCTCGAAGTC 67 2808 2827 33 532636 626 645 Exon 3 CATTGTAGTAGGGAGACCGG 24 2834 2853 34 532637 632 651 Exon 3 CACTCACATTGTAGTAGGGA 49 2840 2859 35 532638 638 657 Exon 3 TCTCATCACTCACATTGTAG 50 2846 2865 36 532639 644 663 Exon 3 AAGAGATCTCATCACTCACA 52 2852 2871 37 532640 650 669 Exon 3 AGTGGAAAGAGATCTCATCA 34 2858 2877 38 532641 656 675 Exon 3 CATAGCAGTGGAAAGAGATC 32 2864 2883 39 532642 662 681 Exon 3 AACCGTCATAGCAGTGGAAA 45 2870 2889 40 532643 668 687 Exon 3 GAGTGTAACCGTCATAGCAG 36 2876 2895 41 532644 674 693 Exon 3 CCCGGAGAGTGTAACCGTCA 30 2882 2901 42 532645 680 699 Exon 3 CAGAGCCCCGGAGAGTGTAA 27 2888 2907 43 532646 686 705 Exon 3 GATTGGCAGAGCCCCGGAGA 20 2894 2913 44 532647 692 711 Exon 3 AGGTGCGATTGGCAGAGCCC 28 2900 2919 45 532648 698 717 Exon 3 CTTGGCAGGTGCGATTGGCA 24 2906 2925 46 532649 704 723 Exon 3 CATTCACTTGGCAGGTGCGA 28 2912 2931 47 532650 729 748 Exon 3 ATCGCTGTCTGCCCACTCCA 44 2937 2956 48 532651 735 754 Exon 3 TCACAGATCGCTGTCTGCCC 44 2943 2962 49 532652 741 760 Exon 3 CCGTTGTCACAGATCGCTGT 27 2949 2968 50 532653 747 766 Exon 3-4 CCCGCTCCGTTGTCACAGAT 28 n/a n/a 51 Junction 532654 753 772 Exon 3-4 CAGTACCCCGCTCCGTTGTC 13 n/a n/a 52 Junction 532655 759 778 Exon 3-4 TTGGAGCAGTACCCCGCTCC 8 n/a n/a 53 Junction 532656 789 808 Exon 4 ACCTTCCTTGTGCCAATGGG 40 3152 3171 54 532657 795 814 Exon 4 CTGCCCACCTTCCTTGTGCC 41 3158 3177 55 532658 818 837 Exon 4 CGCTGTCTTCAAGGCGGTAC 33 3181 3200 56 532659 835 854 Exon 4 GCTGCAGTGGTAGGTGACGC 32 3198 3217 57 532660 841 860 Exon 4 CCCCCGGCTGCAGTGGTAGG 17 3204 3223 58 532661 847 866 Exon 4 GGTAAGCCCCCGGCTGCAGT 28 3210 3229 59 532662 853 872 Exon 4 ACGCAGGGTAAGCCCCCGGC 13 3216 3235 60 532663 859 878 Exon 4 GGAGCCACGCAGGGTAAGCC 33 3222 3241 61 532664 866 885 Exon 4 GCCGCTGGGAGCCACGCAGG 10 3229 3248 62 532665 891 910 Exon 4 CAAGAGCCACCTTCCTGACA 17 3254 3273 63 532666 897 916 Exon 4 CCGCTCCAAGAGCCACCTTC 25 3260 3279 64 532667 903 922 Exon 4 TCCGTCCCGCTCCAAGAGCC 29 3266 3285 65 532668 909 928 Exon 4 GAAGGCTCCGTCCCGCTCCA 14 3272 3291 66 532669 915 934 Exon 4 TGGCAGGAAGGCTCCGTCCC 18 3278 3297 67 532670 921 940 Exon 4-5 GAGTCTTGGCAGGAAGGCTC 20 n/a n/a 68 Junction 532671 927 946 Exon 4-5 ATGAAGGAGTCTTGGCAGGA 14 n/a n/a 69 Junction 532672 956 975 Exon 5 CTTCGGCCACCTCTTGAGGG 45 3539 3558 70 532673 962 981 Exon 5 GGAAAGCTTCGGCCACCTCT 37 3545 3564 71 532674 968 987 Exon 5 AAGACAGGAAAGCTTCGGCC 28 3551 3570 72 532675 974 993 Exon 5 TCAGGGAAGACAGGAAAGCT 16 3557 3576 73 532676 996 1015 Exon 5 TCGACTCCTTCTATGGTCTC 31 3579 3598 74 532677 1033 1052 Exon 5-6 CTTCTGTTGTTCCCCTGGGC 36 n/a n/a 75 Junction 532678 1068 1087 Exon 6 TTCATGGAGCCTGAAGGGTC 19 3752 3771 76 532679 1074 1093 Exon 6 TAGATGTTCATGGAGCCTGA 24 3758 3777 77 532680 1080 1099 Exon 6 ACCAGGTAGATGTTCATGGA 13 3764 3783 78 532681 1086 1105 Exon 6 TCTAGCACCAGGTAGATGTT 20 3770 3789 79 532682 1092 1111 Exon 6 GATCCATCTAGCACCAGGTA 33 3776 3795 80 532683 1098 1117 Exon 6 CTGTCTGATCCATCTAGCAC 44 3782 3801 81 532684 1104 1123 Exon 6 CCAATGCTGTCTGATCCATC 29 3788 3807 82 532685 1129 1148 Exon 6 TTTGGCTCCTGTGAAGTTGC 40 3813 3832 83

TABLE 125 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 ISIS start stop Target % start stop SEQ No site site region Sequence inhibition site site ID NO: 532686 1135 1154 Exon 6 ACACTTTTTGGCTCCTGTGA 91 3819 3838 84 532687 1141 1160 Exon 6 GACTAGACACTTTTTGGCTC 77 3825 3844 85 532688 1147 1166 Exon 6 TAAGTTGACTAGACACTTTT 70 3831 3850 86 532689 1153 1172 Exon 6 CTCAATTAAGTTGACTAGAC 61 3837 3856 87 532690 1159 1178 Exon 6-7 CACCTTCTCAATTAAGTTGA 57 3843 3862 88 Junction 532691 1165 1184 Exon 6-7 ACTTGCCACCTTCTCAATTA 56 n/a n/a 89 Junction 532692 1171 1190 Exon 6-7 ACCATAACTTGCCACCTTCT 56 n/a n/a 90 Junction 532693 1177 1196 Exon 7 CTTCACACCATAACTTGCCA 56 4153 4172 91 532694 1183 1202 Exon 7 TCTTGGCTTCACACCATAAC 55 4159 4178 92 532695 1208 1227 Exon 7 ATGTGGCATATGTCACTAGA 55 4184 4203 93 532696 1235 1254 Exon 7 CAGACACTTTGACCCAAATT 55 4211 4230 94 532697 1298 1317 Exon 7-8 GGTCTTCATAATTGATTTCA 53 n/a n/a 95 Junction 532698 1304 1323 Exon 7-8 ACTTGTGGTCTTCATAATTG 53 n/a n/a 96 Junction 532699 1310 1329 Exon 7-8 ACTTCAACTTGTGGTCTTCA 52 n/a n/a 97 Junction 532700 1316 1335 Exon 8 TCCCTGACTTCAACTTGTGG 52 4609 4628 98 532701 1322 1341 Exon 8 TGTTAGTCCCTGACTTCAAC 52 4615 4634 99 532702 1328 1347 Exon 8 TCTTGGTGTTAGTCCCTGAC 51 4621 4640 100 532703 1349 1368 Exon 8 TGTACACTGCCTGGAGGGCC 51 4642 4661 101 532704 1355 1374 Exon 8 TCATGCTGTACACTGCCTGG 51 4648 4667 102 532705 1393 1412 Exon 8 GTTCCAGCCTTCAGGAGGGA 50 4686 4705 103 532706 1399 1418 Exon 8 GGTGCGGTTCCAGCCTTCAG 50 4692 4711 104 532707 1405 1424 Exon 8 ATGGCGGGTGCGGTTCCAGC 50 4698 4717 105 532708 1411 1430 Exon 8 GATGACATGGCGGGTGCGGT 49 4704 4723 106 532709 1417 1436 Exon 8 GAGGATGATGACATGGCGGG 49 4710 4729 107 532710 1443 1462 Exon 8-9 CCCATGTTGTGCAATCCATC 48 n/a n/a 108 Junction 532711 1449 1468 Exon 9 TCCCCGCCCATGTTGTGCAA 48 5023 5042 109 532712 1455 1474 Exon 9 ATTGGGTCCCCGCCCATGTT 48 5029 5048 110 532713 1461 1480 Exon 9 ACAGTAATTGGGTCCCCGCC 48 5035 5054 111 532714 1467 1486 Exon 9 TCAATGACAGTAATTGGGTC 47 5041 5060 112 532715 1473 1492 Exon 9 ATCTCATCAATGACAGTAAT 47 5047 5066 113 532716 1479 1498 Exon 9 TCCCGGATCTCATCAATGAC 46 5053 5072 114 532717 1533 1552 Exon 9-10 ACATCCAGATAATCCTCCCT 46 n/a n/a 115 Junction 532718 1539 1558 Exon 9-10 ACATAGACATCCAGATAATC 46 n/a n/a 116 Junction 532719 1545 1564 Exon 9-10 CCAAACACATAGACATCCAG 46 n/a n/a 117 Junction 532720 1582 1601 Exon 10 AGCATTGATGTTCACTTGGT 46 5231 5250 118 532721 1588 1607 Exon 10 AGCCAAAGCATTGATGTTCA 45 5237 5256 119 532722 1594 1613 Exon 10 CTTGGAAGCCAAAGCATTGA 45 5243 5262 120 532723 1600 1619 Exon 10 GTCTTTCTTGGAAGCCAAAG 45 5249 5268 121 532724 1606 1625 Exon 10 CTCATTGTCTTTCTTGGAAG 44 5255 5274 122 532725 1612 1631 Exon 10 ATGTTGCTCATTGTCTTTCT 44 5261 5280 123 532726 1618 1637 Exon 10 GAACACATGTTGCTCATTGT 44 5267 5286 124 532727 1624 1643 Exon 10 GACTTTGAACACATGTTGCT 43 5273 5292 125 532728 1630 1649 Exon 10 ATCCTTGACTTTGAACACAT 43 5279 5298 126 532729 1636 1655 Exon 10 TTCCATATCCTTGACTTTGA 43 5285 5304 127 532730 1642 1661 Exon 10 CAGGTTTTCCATATCCTTGA 42 5291 5310 128 532731 1686 1705 Exon 11 CTCAGAGACTGGCTTTCATC 42 5827 5846 129 532732 1692 1711 Exon 11 CAGAGACTCAGAGACTGGCT 42 5833 5852 130 516252 1698 1717 Exon 11 ATGCCACAGAGACTCAGAGA 42 5839 5858 131 532733 1704 1723 Exon 11 CAAACCATGCCACAGAGACT 41 5845 5864 132 532734 1710 1729 Exon 11 TGTTCCCAAACCATGCCACA 41 5851 5870 133 532735 1734 1753 Exon 11 TTGTGGTAATCGGTACCCTT 41 5875 5894 134 532736 1740 1759 Exon 11 GGTTGCTTGTGGTAATCGGT 40 5881 5900 135 532737 1746 1765 Exon 11 TGCCATGGTTGCTTGTGGTA 40 5887 5906 136 532738 1752 1771 Exon 11 TTGGCCTGCCATGGTTGCTT 40 5893 5912 137 532739 1758 1777 Exon 11 GAGATCTTGGCCTGCCATGG 38 5899 5918 138 532740 1803 1822 Exon 12 ACAGCCCCCATACAGCTCTC 38 6082 6101 139 532741 1809 1828 Exon 12 GACACCACAGCCCCCATACA 38 6088 6107 140 532742 1815 1834 Exon 12 TACTCAGACACCACAGCCCC 38 6094 6113 141 532743 1821 1840 Exon 12 ACAAAGTACTCAGACACCAC 37 6100 6119 142 532744 1827 1846 Exon 12 GTCAGCACAAAGTACTCAGA 37 6106 6125 143 532745 1872 1891 Exon 12 TTGATTGAGTGTTCCTTGTC 36 6151 6170 144 532746 1878 1897 Exon 12 CTGACCTTGATTGAGTGTTC 35 6157 6176 145 532747 1909 1928 Exon 13 TATCTCCAGGTCCCGCTTCT 35 6403 6422 146 532748 1967 1986 Exon 13 GAATTCCTGCTTCTTTTTTC 32 6461 6480 147 532749 1973 1992 Exon 13 ATTCAGGAATTCCTGCTTCT 32 6467 6486 148 532750 1979 1998 Exon 13 CATAAAATTCAGGAATTCCT 32 6473 6492 149 532751 1985 2004 Exon 13 CATAGTCATAAAATTCAGGA 31 6479 6498 150 532752 2006 2025 Exon 13 TGAGCTTGATCAGGGCAACG 30 6500 6519 151 532753 2012 2031 Exon 13 TATTCTTGAGCTTGATCAGG 30 6506 6525 152 532754 2048 2067 Exon 13- GACAAATGGGCCTGATAGTC 30 n/a n/a 153 14 Junction 532755 2070 2089 Exon 14 GTTGTTCCCTCGGTGCAGGG 29 6659 6678 154 532756 2076 2095 Exon 14 GCTCGAGTTGTTCCCTCGGT 28 6665 6684 155 532757 2082 2101 Exon 14 CTCAAAGCTCGAGTTGTTCC 28 6671 6690 156 532758 2088 2107 Exon 14 GGAAGCCTCAAAGCTCGAGT 25 6677 6696 157 532759 2094 2113 Exon 14 GTTGGAGGAAGCCTCAAAGC 23 6683 6702 158 532760 2100 2119 Exon 14 GTGGTAGTTGGAGGAAGCCT 23 6689 6708 159 532761 2106 2125 Exon 14 TGGCAAGTGGTAGTTGGAGG 18 6695 6714 160 532762 2112 2131 Exon 14 TGTTGCTGGCAAGTGGTAGT 14 6701 6720 161

TABLE 126 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID ID ID NO: 1 NO: 1 SEQ ID NO: 2 SEQ ISIS start stop Target % NO: 2 start stop ID NO site site Region Sequence inhibition site site NO: 532812 n/a n/a Exon 1 TCCAGCTCACTCCCCTGTTG 19 1593 1612 162 532813 n/a n/a Exon 1 TAAGGATCCAGCTCACTCCC 40 1599 1618 163 532814 n/a n/a Exon 1 CAGAAATAAGGATCCAGCTC 39 1605 1624 164 532815 n/a n/a Exon 1 AGGGACCAGAAATAAGGATC 0 1611 1630 165 532816 n/a n/a Exon 1 CCACTTAGGGACCAGAAATA 27 1617 1636 166 532817 n/a n/a Exon 1 TCCAGGACTCTCCCCTTCAG 39 1682 1701 167 532818 n/a n/a Exon 1 AAGTCCCACCCTTTGCTGCC 15 1707 1726 168 532819 n/a n/a Exon 1 CTGCAGAAGTCCCACCCTTT 26 1713 1732 169 532820 n/a n/a Exon 1 CAGAAACTGCAGAAGTCCCA 8 1719 1738 170 532821 n/a n/a Exon 2- AACCTCTGCACTCTGCCTTC 39 2368 2387 171 Intron 2 532822 n/a n/a Exon 2- CCCTCAAACCTCTGCACTCT 3 2374 2393 172 Intron 2 532823 n/a n/a Exon 2- TCATTGCCCTCAAACCTCTG 19 2380 2399 173 Intron 2 532824 n/a n/a Intron 2 CCACACTCATTGCCCTCAAA 37 2386 2405 174 532825 n/a n/a Intron 2 CACTGCCCACACTCATTGCC 23 2392 2411 175 532826 n/a n/a Intron 2 TTAGGCCACTGCCCACACTC 15 2398 2417 176 532827 n/a n/a Intron 2 CTAGTCCTGACCTTGCTGCC 28 2436 2455 177 532828 n/a n/a Intron 2 CTCATCCTAGTCCTGACCTT 25 2442 2461 178 532829 n/a n/a Intron 2 CCTAGTCTCATCCTAGTCCT 23 2448 2467 179 532830 n/a n/a Intron 2 ACCCTGCCTAGTCTCATCCT 30 2454 2473 180 532831 n/a n/a Intron 2 CTTGTCACCCTGCCTAGTCT 34 2460 2479 181 532832 n/a n/a Intron 2 GCCCACCTTGTCACCCTGCC 36 2466 2485 182 532833 n/a n/a Intron 2 CCTAAAACTGCTCCTACTCC 9 2492 2511 183 532834 n/a n/a Intron 4 GAGTCAGAAATGAGGTCAAA 19 3494 3513 184 532835 n/a n/a Intron CCCTACTCCCATTTCACCTT 16 5971 5990 185 11 532836 n/a n/a Intron 8- TGTTGTGCAATCCTGCAGAA 25 5013 5032 186 Exon 9 532837 n/a n/a Intron 1 AAAGGCTGATGAAGCCTGGC 18 2123 2142 187 532838 n/a n/a Intron 7 CCTTTGACCACAAAGTGGCC 21 4461 4480 188 532839 n/a n/a Intron AGGTACCACCTCTTTGTGGG 29 6362 6381 189 12 532840 n/a n/a Intron 1- TGGTGGTCACACCTGAAGAG 34 2143 2162 190 Exon 2 532763 2133 2152 Exon GCAGGGAGCAGCTCTTCCTT 40 n/a n/a 191 14-15 Junction 532764 2139 2158 Exon 15 TCCTGTGCAGGGAGCAGCTC 28 6927 6946 192 532765 2145 2164 Exon 15 TTGATATCCTGTGCAGGGAG 41 6933 6952 193 532766 2151 2170 Exon 15 AGAGCTTTGATATCCTGTGC 36 6939 6958 194 532767 2157 2176 Exon 15 ACAAACAGAGCTTTGATATC 33 6945 6964 195 532768 2163 2182 Exon 15 TCAGACACAAACAGAGCTTT 41 6951 6970 196 532769 2169 2188 Exon 15 TCCTCCTCAGACACAAACAG 49 6957 6976 197 532770 2193 2212 Exon 15 ACCTCCTTCCGAGTCAGCTT 61 6981 7000 198 532771 2199 2218 Exon 15 ATGTAGACCTCCTTCCGAGT 39 6987 7006 199 532772 2205 2224 Exon 15 TTCTTGATGTAGACCTCCTT 30 6993 7012 200 532773 2211 2230 Exon 15 TCCCCATTCTTGATGTAGAC 31 6999 7018 201 532774 2217 2236 Exon TTCTTATCCCCATTCTTGAT 36 n/a n/a 202 15-16 Junction 532775 2223 2242 Exon CTGCCTTTCTTATCCCCATT 56 n/a n/a 203 15-16 Junction 532776 2229 2248 Exon TCACAGCTGCCTTTCTTATC 33 n/a n/a 204 15-16 Junction 532777 2235 2254 Exon 16 TCTCTCTCACAGCTGCCTTT 38 7119 7138 205 532778 2241 2260 Exon 16 TGAGCATCTCTCTCACAGCT 51 7125 7144 206 532779 2247 2266 Exon 16 GCATATTGAGCATCTCTCTC 39 7131 7150 207 532780 2267 2286 Exon 16 TGACTTTGTCATAGCCTGGG 56 7151 7170 208 532781 2273 2292 Exon 16 TGTCCTTGACTTTGTCATAG 36 7157 7176 209 532782 2309 2328 Exon 16 CAGTACAAAGGAACCGAGGG 30 7193 7212 210 532783 2315 2334 Exon 16 CTCCTCCAGTACAAAGGAAC 21 7199 7218 211 532784 2321 2340 Exon 16 GACTCACTCCTCCAGTACAA 31 7205 7224 212 532785 2327 2346 Exon 16 CATAGGGACTCACTCCTCCA 30 7211 7230 213 532786 2333 2352 Exon 16 GGTCAGCATAGGGACTCACT 31 7217 7236 214 532787 2352 2371 Exon TCACCTCTGCAAGTATTGGG 42 7236 7255 215 16-17 Junction 532788 2358 2377 Exon CCAGAATCACCTCTGCAAGT 32 n/a n/a 216 16-17 Junction 532789 2364 2383 Exon GGGCCGCCAGAATCACCTCT 35 n/a n/a 217 16-17 Junction 532790 2382 2401 Exon 17 CTCTTGTGAACTATCAAGGG 33 7347 7366 218 532791 2388 2407 Exon 17 CGACTTCTCTTGTGAACTAT 52 7353 7372 219 532792 2394 2413 Exon 17 ATGAAACGACTTCTCTTGTG 16 7359 7378 220 532793 2400 2419 Exon ACTTGAATGAAACGACTTCT 45 7365 7384 221 17-18 Junction 532794 2406 2425 Exon ACACCAACTTGAATGAAACG 18 n/a n/a 222 17-18 Junction 532795 2427 2446 Exon 18 TCCACTACTCCCCAGCTGAT 30 7662 7681 223 532796 2433 2452 Exon 18 CAGACATCCACTACTCCCCA 38 7668 7687 224 532797 2439 2458 Exon 18 TTTTTGCAGACATCCACTAC 35 7674 7693 225 532798 2445 2464 Exon 18 TTCTGGTTTTTGCAGACATC 45 7680 7699 226 532799 2451 2470 Exon 18 TGCCGCTTCTGGTTTTTGCA 47 7686 7705 227 532800 2457 2476 Exon 18 TGCTTTTGCCGCTTCTGGTT 61 7692 7711 228 532801 2463 2482 Exon 18 GGTACCTGCTTTTGCCGCTT 47 7698 7717 229 532802 2469 2488 Exon 18 TGAGCAGGTACCTGCTTTTG 31 7704 7723 230 532803 2517 2536 Exon 18 TTCAGCCAGGGCAGCACTTG 41 7752 7771 231 532804 2523 2542 Exon 18 TTCTCCTTCAGCCAGGGCAG 44 7758 7777 232 532805 2529 2548 Exon 18 TGGAGTTTCTCCTTCAGCCA 46 7764 7783 233 532806 2535 2554 Exon 18 TCATCTTGGAGTTTCTCCTT 49 7770 7789 234 532807 2541 2560 Exon 18 AAATCCTCATCTTGGAGTTT 30 7776 7795 235 532808 2547 2566 Exon 18 AAACCCAAATCCTCATCTTG 20 7782 7801 236 532809 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 65 7806 7825 237 532810 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 74 7812 7831 238 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 96 7834 7853 239

TABLE 127 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target start stop % ID NO site site region Sequence site site inhibition NO: 532841 n/a n/a Intron 6- AACTTGCCACCTGTGGGTGA 4142 4161 11 240 Exon 7 532842 n/a n/a Exon 15- TCACCTTATCCCCATTCTTG 7007 7026 16 241 Intron 15 532843 n/a n/a Intron 11 TCAACTTTCACAAACCACCA 6015 6034 19 242 532844 n/a n/a Intron 16- CCGCCAGAATCACCTGCAAG 7326 7345 33 243 Exon 17 532845 n/a n/a Intron 10 AGGAGGAATGAAGAAGGCTT 5431 5450 29 244 532846 n/a n/a Intron 13 GCCTTTCCTCAGGGATCTGG 6561 6580 26 245 532847 n/a n/a Intron 4 AAATGTCTGGGAGTGTCAGG 3477 3496 18 246 532848 n/a n/a Intron 15 GCCTAGAGTGCCTCCTTAGG 7038 7057 20 247 532849 n/a n/a Intron 17 GGCATCTCCCCAGATAGGAA 7396 7415 16 248 532850 n/a n/a Intron 6 AGGGAGCTAGTCCTGGAAGA 3906 3925 14 249 532851 n/a n/a Intron 1- ACACCTGAAGAGAAAGGCTG 2135 2154 6 250 Exon 2 532852 n/a n/a Intron 7 CCCTTTGACCACAAAGTGGC 4462 4481 25 251 532853 n/a n/a Intron 7 GCCCTCAAGGTAGTCTCATG 4354 4373 26 252 532854 n/a n/a Intron 6 AAGGGAAGGAGGACAGAATA 3977 3996 18 253 532855 n/a n/a Intron 1 AAAGGCCAAGGAGGGATGCT 2099 2118 9 254 532856 n/a n/a Exon 8- AGAGGTCCCTTCTGACCATC 4736 4755 4 255 Intron 8 532857 n/a n/a Intron 8 GCTGGGACAGGAGAGAGGTC 4749 4768 0 256 532858 n/a n/a Intron 4 TCAAATGTCTGGGAGTGTCA 3479 3498 13 257 532859 n/a n/a Intron 10 AGAAGGAGAATGTGCTGAAA 5801 5820 20 258 532860 n/a n/a Intron 17 TGCTGACCACTTGGCATCTC 7408 7427 20 259 532861 n/a n/a Intron 11 CAACTTTCACAAACCACCAT 6014 6033 18 260 532862 n/a n/a Intron 10 AGCTCTGTGATTCTAAGGTT 5497 5516 15 261 532863 n/a n/a Intron 6- CCACCTGTGGGTGAGGAGAA 4136 4155 16 262 Exon 7 532864 n/a n/a Exon 17- GAGGACTCACTTGAATGAAA 7373 7392 21 263 Intron 17 532865 n/a n/a Intron 6 TGGAATGATCAGGGAGCTAG 3916 3935 30 264 532866 n/a n/a Intron 5 GTCCCTTCTCCATTTTCCCC 3659 3678 26 265 532867 n/a n/a Intron 7 TCAACTTTTTAAGTTAATCA 4497 4516 14 266 532868 n/a n/a Intron 6 GGGTGAGGAGAACAAGGCGC 4128 4147 21 267 532869 n/a n/a Intron 7 CTTCCAAGCCATCTTTTAAC 4553 4572 5 268 532870 n/a n/a Exon 17- AGGACTCACTTGAATGAAAC 7372 7391 18 269 Intron 17 532871 n/a n/a Intron 10 TTCCAGGCAACTAGAGCTTC 5412 5431 15 270 532872 n/a n/a Exon 1 CAGAGTCCAGCCACTGTTTG 1557 1576 13 271 532873 n/a n/a Intron 17- CCAACCTGCAGAGGCAGTGG 7638 7657 23 272 Exon 18 532874 n/a n/a Intron 16 TGCAAGGAGAGGAGAAGCTG 7312 7331 10 273 532875 n/a n/a Exon 9- CTAGGCAGGTTACTCACCCA 5120 5139 21 274 Intron 9 532876 n/a n/a Intron 6- CACCATAACTTGCCACCTGT 4148 4167 41 275 Exon 7 532877 n/a n/a Intron 12 TAGGTACCACCTCTTTGTGG 6363 6382 27 276 532878 n/a n/a Intron 11 CTTGACCTCACCTCCCCCAA 5954 5973 13 277 532879 n/a n/a Intron 12 CCACCTCTTTGTGGGCAGCT 6357 6376 33 278 532880 n/a n/a Intron 11 TTCACAAACCACCATCTCTT 6009 6028 8 279 532881 n/a n/a Exon 3- TTCTCACCTCCGTTGTCACA 2958 2977 17 280 Intron 3 532882 n/a n/a Intron 12 GAAAGTGGGAGGTGTTGCCT 6225 6244 19 281 532883 n/a n/a Intron 1 ACAGCAGGAAGGGAAGGTTA 2075 2094 34 282 532884 n/a n/a Intron 17 CATGCTGACCACTTGGCATC 7410 7429 18 283 532885 n/a n/a Exon 4- GGTCACCTTGGCAGGAAGGC 3286 3305 0 284 Intron 4 532886 n/a n/a Intron 8 GTATAGTGTTACAAGTGGAC 4804 4823 13 285 532887 n/a n/a Intron 7 GGACTTCCCTTTGACCACAA 4468 4487 18 286 532888 n/a n/a Intron 11 TCACCTTGACCTCACCTCCC 5958 5977 20 287 532889 n/a n/a Intron 15 TAGAGTGCCTCCTTAGGATG 7035 7054 27 288 532890 n/a n/a Intron 7 TGACTTCAACTTGTGGTCTG 4605 4624 16 289 532891 n/a n/a Intron 10 CAGAGAAGGAGAATGTGCTG 5804 5823 25 290 532892 n/a n/a Intron 14- AGGGAGCAGCTCTTCCTCTG 6919 6938 47 291 Exon 15 532893 n/a n/a Intron 5- TGTTCCCCTGGGTGCCAGGA 3710 3729 24 292 Exon 6 532894 n/a n/a Intron 10 GGCCTGGCTGTTTTCAAGCC 5612 5631 15 293 532895 n/a n/a Intron 10- GACTGGCTTTCATCTGGCAG 5821 5840 25 294 Exon 11 532896 n/a n/a Intron 10 GAAGGCTTTCCAGGCAACTA 5419 5438 19 295 532897 n/a n/a Exon 17- TCACTTGAATGAAACGACTT 7367 7386 11 296 Intron 17 532898 n/a n/a Intron 1 GGCCCCAAAAGGCCAAGGAG 2106 2125 5 297 532899 n/a n/a Intron 16- AATCACCTGCAAGGAGAGGA 7319 7338 19 298 Exon 17 532900 n/a n/a Intron 12 GACCTTCAGTTGCATCCTTA 6183 6202 25 299 532901 n/a n/a Intron 1 TGATGAAGCCTGGCCCCAAA 2117 2136 0 300 532902 n/a n/a Intron 12 TAGAAAGTGGGAGGTGTTGC 6227 6246 0 301 532903 n/a n/a Intron 12 CCCATCCCTGACTGGTCTGG 6295 6314 14 302 532904 n/a n/a Intron 8 CCATGGGTATAGTGTTACAA 4810 4829 13 303 532905 n/a n/a Intron 2 GTGTTCTCTTGACTTCCAGG 2586 2605 23 304 532906 n/a n/a Intron 13 GGCCTGCTCCTCACCCCAGT 6597 6616 27 305 532907 n/a n/a Intron 10 GAGGCCTGGCTGTTTTCAAG 5614 5633 32 306 532908 n/a n/a Exon 1 GACTCTCCCCTTCAGTACCT 1677 1696 16 307 532909 n/a n/a Intron 8 CATGGGTATAGTGTTACAAG 4809 4828 10 308 532910 n/a n/a Intron 10 GAAGGAGAATGTGCTGAAAA 5800 5819 0 309 532911 n/a n/a Intron 7 TCACCTGGTCTTCCAAGCCA 4562 4581 0 310 532912 n/a n/a Intron 17 CTCCCCAGATAGGAAAGGGA 7391 7410 0 311 532913 n/a n/a Exon 17- GGACTCACTTGAATGAAACG 7371 7390 0 312 Intron 17 532914 n/a n/a Intron 16- GGCCGCCAGAATCACCTGCA 7328 7347 30 313 Exon 17 532915 n/a n/a Exon 17- CTCACTTGAATGAAACGACT 7368 7387 22 314 Intron 17 532916 n/a n/a Intron 13 CTTTCCCAGCCTTTCCTCAG 6569 6588 28 315 532918 n/a n/a Intron 12 AGAAAGTGGGAGGTGTTGCC 6226 6245 3 316 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 7839 7858 90 317

TABLE 128 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target start stop % ID NO site site region Sequence site site inhibition NO: 532919 n/a n/a Exon 1 CCAGGACTCTCCCCTTCAGT 1681 1700 4 318 532920 n/a n/a Intron 6 AGGGAAGGAGGACAGAATAG 3976 3995 25 319 532921 n/a n/a Intron 4 GAAATGAGGTCAAATGTCTG 3488 3507 30 320 532922 n/a n/a Intron 4 GGAGAGTCAGAAATGAGGTC 3497 3516 25 321 532923 n/a n/a Intron 12 GTAGAAAGTGGGAGGTGTTG 6228 6247 26 322 532924 n/a n/a Intron 10 TAGAAAGATCTCTGAAGTGC 5521 5540 24 323 532925 n/a n/a Intron 13 CTGCTCCTCACCCCAGTCCT 6594 6613 26 324 532926 n/a n/a Intron 11 CTACTGGGATTCTGTGCTTA 5927 5946 30 325 532927 n/a n/a Intron 1 CCCAAAAGGCCAAGGAGGGA 2103 2122 13 326 532928 n/a n/a Intron 17 TGACCACTTGGCATCTCCCC 7405 7424 27 327 532929 n/a n/a Intron 16- CCTGCAAGGAGAGGAGAAGC 7314 7333 29 328 Exon 17 532930 n/a n/a Exon 16- CTCTCACCTCTGCAAGTATT 7239 7258 44 329 Intron 16 532931 n/a n/a Intron 1 CCCCAAAAGGCCAAGGAGGG 2104 2123 21 330 532932 n/a n/a Intron 7 GTCTTCCAAGCCATCTTTTA 4555 4574 20 331 532933 n/a n/a Intron 8 GTTACAAGTGGACTTAAGGG 4797 4816 30 332 532934 n/a n/a Intron 8- CCCATGTTGTGCAATCCTGC 5017 5036 30 333 Exon 9 532935 n/a n/a Intron 15 GAGGTGGGAAGCATGGAGAA 7091 7110 17 334 532936 n/a n/a Intron 14 TGCTCCCACCACTGTCATCT 6874 6893 21 335 532937 n/a n/a Exon 9- AGGCAGGTTACTCACCCAGA 5118 5137 18 336 Intron 9 532938 n/a n/a Intron 11 TACTGGGATTCTGTGCTTAC 5926 5945 15 337 532939 n/a n/a Intron 13 GCCTTTCCCAGCCTTTCCTC 6571 6590 27 338 532940 n/a n/a Intron 8- GTGCAATCCTGCAGAAGAGA 5009 5028 21 339 Exon 9 532941 n/a n/a Intron 8 ACAGGAGAGAGGTCCCTTCT 4743 4762 20 340 532942 n/a n/a Intron 10 CCCAAAAGGAGAAAGGGAAA 5717 5736 14 341 532943 n/a n/a Intron 2 AAGCCCAGGGTAAATGCTTA 2557 2576 32 342 532944 n/a n/a Intron 1 GATGAAGCCTGGCCCCAAAA 2116 2135 22 343 532945 n/a n/a Intron 10 TGGCAGAGAAGGAGAATGTG 5807 5826 22 344 532946 n/a n/a Intron 13 TTCCCAGCCTTTCCTCAGGG 6567 6586 35 345 532947 n/a n/a Intron 10 GGCAGAGAAGGAGAATGTGC 5806 5825 30 346 532948 n/a n/a Intron 10 ACAGTGCCAGGAAACAAGAA 5471 5490 25 347 532949 n/a n/a Exon 9- TAGGCAGGTTACTCACCCAG 5119 5138 22 348 Intron 9 532950 n/a n/a Intron 2 TTCTCTTGACTTCCAGGGCT 2583 2602 22 349 532951 n/a n/a Intron 13 CCTGCTCCTCACCCCAGTCC 6595 6614 16 350 532953 n/a n/a Intron 7 TCCCACTAACCTCCATTGCC 4422 4441 14 351 532954 n/a n/a Intron 7 TTCCCTTTGACCACAAAGTG 4464 4483 16 352 532955 n/a n/a Intron 9 CTGGGTCCTAGGCAGGTTAC 5127 5146 30 353 532956 n/a n/a Intron 10 TCCAGGCAACTAGAGCTTCA 5411 5430 20 354 532957 n/a n/a Intron 8- GCCCATGTTGTGCAATCCTG 5018 5037 45 355 Exon 9 532958 n/a n/a Intron 7 GGTTCCCACTAACCTCCATT 4425 4444 18 356 532959 n/a n/a Intron 3 AGGTAGAGAGCAAGAGTTAC 3052 3071 26 357 532960 n/a n/a Intron 7 CCACTAACCTCCATTGCCCA 4420 4439 10 358 532961 n/a n/a Intron 11 TCACAAACCACCATCTCTTA 6008 6027 40 359 532962 n/a n/a Exon 9- TACTCACCCAGATAATCCTC 5110 5129 27 360 Intron 9 532963 n/a n/a Intron 13 TGCTCCTCACCCCAGTCCTC 6593 6612 24 361 532964 n/a n/a Intron 15- TCTCACAGCTGCCTTTCTGT 7115 7134 25 362 Exon 16 532965 n/a n/a Exon 17- GAAAGGGAGGACTCACTTGA 7379 7398 11 363 Intron 17 532966 n/a n/a Intron 7 CCATCTTTTAACCCCAGAGA 4545 4564 18 364 532967 n/a n/a Intron 13 TCCTCACCCCAGTCCTCCAG 6590 6609 27 365 532968 n/a n/a Intron 10 CTGGCAGAGAAGGAGAATGT 5808 5827 15 366 532969 n/a n/a Intron 17 TCTCCCCAGATAGGAAAGGG 7392 7411 23 367 532970 n/a n/a Intron 14 ACTTCAGCTGCTCCCACCAC 6882 6901 18 368 532971 n/a n/a Intron 1 GACAGCAGGAAGGGAAGGTT 2076 2095 13 369 532972 n/a n/a Intron 13- GGAGACAAATGGGCCTATAA 6640 6659 33 370 Exon 14 532973 n/a n/a Intron 14 CTGCTCCCACCACTGTCATC 6875 6894 11 371 532974 n/a n/a Intron 10 AGGAATGAAGAAGGCTTTCC 5428 5447 21 372 532975 n/a n/a Intron 14 GGGATCTCATCCTTATCCTC 6741 6760 31 373 532976 n/a n/a Intron 9 GTGCTGGGTCCTAGGCAGGT 5130 5149 16 374 532977 n/a n/a Intron 1 CAAAAGGCCAAGGAGGGATG 2101 2120 14 375 532978 n/a n/a Intron 17 CCATGCTGACCACTTGGCAT 7411 7430 20 376 532979 n/a n/a Intron 8 GGAGGCTGGGACAGGAGAGA 4753 4772 25 377 532980 n/a n/a Intron 14- GGAGCAGCTCTTCCTCTGGA 6917 6936 36 378 Exon 15 532981 n/a n/a Exon 3- TCTCACCTCCGTTGTCACAG 2957 2976 20 379 Intron 3 532982 n/a n/a Intron 13 CAGTCCTCCAGCCTTTCCCA 6581 6600 21 380 532983 n/a n/a Intron 13 AGTCCTCCAGCCTTTCCCAG 6580 6599 22 381 532984 n/a n/a Intron 4- TGAAGGAGTCTGGGAGAGTC 3509 3528 12 382 Exon 5 532985 n/a n/a Intron 16- CAGAATCACCTGCAAGGAGA 7322 7341 20 383 Exon 17 532986 n/a n/a Exon 17- TAGGAAAGGGAGGACTCACT 7382 7401 3 384 Intron 17 532987 n/a n/a Exon 4- ACCTTGGCAGGAAGGCTCCG 3282 3301 12 385 Intron 4 532988 n/a n/a Intron 13- GAGACAAATGGGCCTATAAA 6639 6658 15 386 Exon 14 532989 n/a n/a Intron 1 CTGAAGAGAAAGGCTGATGA 2131 2150 17 387 532990 n/a n/a Intron 6 AATGATCAGGGAGCTAGTCC 3913 3932 30 388 532991 n/a n/a Intron 17 CTTAGCTGACCTAAAGGAAT 7557 7576 22 389 532992 n/a n/a Intron 8 TGGGTATAGTGTTACAAGTG 4807 4826 17 390 532993 n/a n/a Intron 1 TGAAGAGAAAGGCTGATGAA 2130 2149 19 391 532994 n/a n/a Intron 8 GTGTTACAAGTGGACTTAAG 4799 4818 25 392 532995 n/a n/a Intron 6 ACCTGTGGGTGAGGAGAACA 4134 4153 24 393 532996 n/a n/a Exon 9- TCACCCAGATAATCCTCCCT 5107 5126 36 394 Intron 9 532952 2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 7843 7862 90 395

Example 116: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3460_MGB (forward sequence CGAAGCAGCTCAATGAAATCAA, designated herein as SEQ ID NO: 813; reverse sequence TGCCTGGAGGGCCTTCTT, designated herein as SEQ ID NO: 814; probe sequence AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815) was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 129 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site NO: 532686 1135 1154 Exon 6 ACACTTTTTGGCTCCTGTGA 48 3819 3838 84 532687 1141 1160 Exon 6 GACTAGACACTTTTTGGCTC 63 3825 3844 85 532688 1147 1166 Exon 6 TAAGTTGACTAGACACTTTT 47 3831 3850 86 532689 1153 1172 Exon 6 CTCAATTAAGTTGACTAGAC 57 3837 3856 87 532690 1159 1178 Exon 6-7 CACCTTCTCAATTAAGTTGA 49 3843 3862 88 Junction 532691 1165 1184 Exon 6-7 ACTTGCCACCTTCTCAATTA 33 n/a n/a 89 Junction 532692 1171 1190 Exon 6-7 ACCATAACTTGCCACCTTCT 67 n/a n/a 90 Junction 532693 1177 1196 Exon 7 CTTCACACCATAACTTGCCA 56 4153 4172 91 532694 1183 1202 Exon 7 TCTTGGCTTCACACCATAAC 50 4159 4178 92 532695 1208 1227 Exon 7 ATGTGGCATATGTCACTAGA 53 4184 4203 93 532696 1235 1254 Exon 7 CAGACACTTTGACCCAAATT 52 4211 4230 94 532697 1298 1317 Exon 7-8 GGTCTTCATAATTGATTTCA 59 n/a n/a 95 Junction 532698 1304 1323 Exon 7-8 ACTTGTGGTCTTCATAATTG 52 n/a n/a 96 Junction 532699 1310 1329 Exon 7-8 ACTTCAACTTGTGGTCTTCA 85 n/a n/a 97 Junction 532700 1316 1335 Exon 8 TCCCTGACTTCAACTTGTGG 96 4609 4628 98 532701 1322 1341 Exon 8 TGTTAGTCCCTGACTTCAAC 56 4615 4634 99 532702 1328 1347 Exon 8 TCTTGGTGTTAGTCCCTGAC 86 4621 4640 100 532703 1349 1368 Exon 8 TGTACACTGCCTGGAGGGCC 35 4642 4661 101 532704 1355 1374 Exon 8 TCATGCTGTACACTGCCTGG 12 4648 4667 102 532705 1393 1412 Exon 8 GTTCCAGCCTTCAGGAGGGA 27 4686 4705 103 532706 1399 1418 Exon 8 GGTGCGGTTCCAGCCTTCAG 67 4692 4711 104 532707 1405 1424 Exon 8 ATGGCGGGTGCGGTTCCAGC 26 4698 4717 105 532708 1411 1430 Exon 8 GATGACATGGCGGGTGCGGT 28 4704 4723 106 532709 1417 1436 Exon 8 GAGGATGATGACATGGCGGG 6 4710 4729 107 532710 1443 1462 Exon 8-9 CCCATGTTGTGCAATCCATC 35 n/a n/a 108 Junction 532711 1449 1468 Exon 9 TCCCCGCCCATGTTGTGCAA 28 5023 5042 109 532712 1455 1474 Exon 9 ATTGGGTCCCCGCCCATGTT 19 5029 5048 110 532713 1461 1480 Exon 9 ACAGTAATTGGGTCCCCGCC 29 5035 5054 111 532714 1467 1486 Exon 9 TCAATGACAGTAATTGGGTC 49 5041 5060 112 532715 1473 1492 Exon 9 ATCTCATCAATGACAGTAAT 45 5047 5066 113 532716 1479 1498 Exon 9 TCCCGGATCTCATCAATGAC 54 5053 5072 114 532717 1533 1552 Exon 9- ACATCCAGATAATCCTCCCT 22 n/a n/a 115 10 Junction 532718 1539 1558 Exon 9- ACATAGACATCCAGATAATC 8 n/a n/a 116 10 Junction 532719 1545 1564 Exon 9- CCAAACACATAGACATCCAG 30 n/a n/a 117 10 Junction 532720 1582 1601 Exon 10 AGCATTGATGTTCACTTGGT 62 5231 5250 118 532721 1588 1607 Exon 10 AGCCAAAGCATTGATGTTCA 46 5237 5256 119 532722 1594 1613 Exon 10 CTTGGAAGCCAAAGCATTGA 35 5243 5262 120 532723 1600 1619 Exon 10 GTCTTTCTTGGAAGCCAAAG 43 5249 5268 121 532724 1606 1625 Exon 10 CTCATTGTCTTTCTTGGAAG 40 5255 5274 122 532725 1612 1631 Exon 10 ATGTTGCTCATTGTCTTTCT 49 5261 5280 123 532726 1618 1637 Exon 10 GAACACATGTTGCTCATTGT 68 5267 5286 124 532727 1624 1643 Exon 10 GACTTTGAACACATGTTGCT 54 5273 5292 125 532728 1630 1649 Exon 10 ATCCTTGACTTTGAACACAT 61 5279 5298 126 532729 1636 1655 Exon 10 TTCCATATCCTTGACTTTGA 55 5285 5304 127 532730 1642 1661 Exon 10 CAGGTTTTCCATATCCTTGA 51 5291 5310 440 532731 1686 1705 Exon 10- CTCAGAGACTGGCTTTCATC 41 5827 5846 129 11 Junction 532732 1692 1711 Exon 11 CAGAGACTCAGAGACTGGCT 59 5833 5852 130 516252 1698 1717 Exon 11 ATGCCACAGAGACTCAGAGA 57 5839 5858 131 532733 1704 1723 Exon 11 CAAACCATGCCACAGAGACT 34 5845 5864 132 532734 1710 1729 Exon 11 TGTTCCCAAACCATGCCACA 51 5851 5870 133 532735 1734 1753 Exon 11 TTGTGGTAATCGGTACCCTT 50 5875 5894 134 532736 1740 1759 Exon 11 GGTTGCTTGTGGTAATCGGT 64 5881 5900 135 532737 1746 1765 Exon 11 TGCCATGGTTGCTTGTGGTA 40 5887 5906 136 532738 1752 1771 Exon 11 TTGGCCTGCCATGGTTGCTT 49 5893 5912 137 532739 1758 1777 Exon 11 GAGATCTTGGCCTGCCATGG 47 5899 5918 138 532740 1803 1822 Exon 12 ACAGCCCCCATACAGCTCTC 48 6082 6101 139 532741 1809 1828 Exon 12 GACACCACAGCCCCCATACA 40 6088 6107 140 532742 1815 1834 Exon 12 TACTCAGACACCACAGCCCC 33 6094 6113 141 532743 1821 1840 Exon 12 ACAAAGTACTCAGACACCAC 39 6100 6119 142 532744 1827 1846 Exon 12 GTCAGCACAAAGTACTCAGA 45 6106 6125 143 532745 1872 1891 Exon 12 TTGATTGAGTGTTCCTTGTC 42 6151 6170 144 532746 1878 1897 Exon 12 CTGACCTTGATTGAGTGTTC 53 6157 6176 145 532747 1909 1928 Exon 13 TATCTCCAGGTCCCGCTTCT 31 6403 6422 146 532748 1967 1986 Exon 13 GAATTCCTGCTTCTTTTTTC 30 6461 6480 147 532749 1973 1992 Exon 13 ATTCAGGAATTCCTGCTTCT 40 6467 6486 148 532750 1979 1998 Exon 13 CATAAAATTCAGGAATTCCT 45 6473 6492 149 532751 1985 2004 Exon 13 CATAGTCATAAAATTCAGGA 43 6479 6498 150 532752 2006 2025 Exon 13 TGAGCTTGATCAGGGCAACG 61 6500 6519 151 532753 2012 2031 Exon 13 TATTCTTGAGCTTGATCAGG 47 6506 6525 152 532754 2048 2067 Exon 13- GACAAATGGGCCTGATAGTC 35 n/a n/a 153 14 Junction 532755 2070 2089 Exon 14 GTTGTTCCCTCGGTGCAGGG 43 6659 6678 154 532756 2076 2095 Exon 14 GCTCGAGTTGTTCCCTCGGT 51 6665 6684 155 532757 2082 2101 Exon 14 CTCAAAGCTCGAGTTGTTCC 36 6671 6690 156 532758 2088 2107 Exon 14 GGAAGCCTCAAAGCTCGAGT 54 6677 6696 157 532759 2094 2113 Exon 14 GTTGGAGGAAGCCTCAAAGC 52 6683 6702 158 532760 2100 2119 Exon 14 GTGGTAGTTGGAGGAAGCCT 22 6689 6708 159 532761 2106 2125 Exon 14 TGGCAAGTGGTAGTTGGAGG 34 6695 6714 160 532762 2112 2131 Exon 14 TGTTGCTGGCAAGTGGTAGT 52 6701 6720 161

Example 117: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 5,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene.

TABLE 130 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 SEQ ID SEQ ID SEQ ISIS NO: 1 NO: 1 stop Target % ID NO start site site region Sequence inhibition NO: 588570 150 169 Exon 1 TGGTCACATTCCCTTCCCCT 54 396 588571 152 171 Exon 1 CCTGGTCACATTCCCTTCCC 63 397 532614 154 173 Exon 1 GACCTGGTCACATTCCCTTC 64 12 588572 156 175 Exon 1 TAGACCTGGTCACATTCCCT 62 398 588573 158 177 Exon 1 CCTAGACCTGGTCACATTCC 53 399 588566 2189 2208 Exon 15 CCTTCCGAGTCAGCTTTTTC 60 400 588567 2191 2210 Exon 15 CTCCTTCCGAGTCAGCTTTT 61 401 532770 2193 2212 Exon 15 ACCTCCTTCCGAGTCAGCTT 77 198 588568 2195 2214 Exon 15 AGACCTCCTTCCGAGTCAGC 72 402 588569 2197 2216 Exon 15 GTAGACCTCCTTCCGAGTCA 46 403 588574 2453 2472 Exon 18 TTTGCCGCTTCTGGTTTTTG 46 404 588575 2455 2474 Exon 18 CTTTTGCCGCTTCTGGTTTT 41 405 532800 2457 2476 Exon 18 TGCTTTTGCCGCTTCTGGTT 69 228 588576 2459 2478 Exon 18 CCTGCTTTTGCCGCTTCTGG 61 406 588577 2461 2480 Exon 18 TACCTGCTTTTGCCGCTTCT 51 407 516350 2550 2569 Exon 18 AGAAAACCCAAATCCTCATC 71 408 588509 2551 2570 Exon 18 TAGAAAACCCAAATCCTCAT 58 409 588510 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 57 410 588511 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 57 411 588512 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 44 412 588513 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 37 413 588514 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC 50 414 588515 2557 2576 Exon 18 CCCTTATAGAAAACCCAAAT 45 415 588516 2558 2577 Exon 18 CCCCTTATAGAAAACCCAAA 60 416 588517 2559 2578 Exon 18 ACCCCTTATAGAAAACCCAA 67 417 588518 2560 2579 Exon 18 AACCCCTTATAGAAAACCCA 57 418 588519 2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 61 419 588520 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 27 420 588521 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 25 421 588522 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 36 422 588523 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 36 423 588524 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA 46 424 588525 2567 2586 Exon 18 AGCAGGAAACCCCTTATAGA 38 425 588526 2568 2587 Exon 18 CAGCAGGAAACCCCTTATAG 47 426 588527 2569 2588 Exon 18 CCAGCAGGAAACCCCTTATA 68 427 588528 2570 2589 Exon 18 TCCAGCAGGAAACCCCTTAT 63 428 532809 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 85 237 588529 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 76 429 588530 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 74 430 588531 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 75 431 588532 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 73 432 588533 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC 82 433 532810 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 88 238 588534 2578 2597 Exon 18 CGCCCCTGTCCAGCAGGAAA 86 434 588535 2579 2598 Exon 18 ACGCCCCTGTCCAGCAGGAA 86 435 588536 2580 2599 Exon 18 CACGCCCCTGTCCAGCAGGA 93 436 588537 2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 92 437 588538 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 94 438 588539 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 96 439 588540 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 88 440 588541 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 79 441 588542 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA 83 442 588543 2587 2606 Exon 18 TCAATCCCACGCCCCTGTCC 86 443 588544 2588 2607 Exon 18 TTCAATCCCACGCCCCTGTC 90 444 588545 2589 2608 Exon 18 ATTCAATCCCACGCCCCTGT 92 445 588546 2590 2609 Exon 18 AATTCAATCCCACGCCCCTG 92 446 588547 2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 88 447 588548 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 93 448 588549 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 88 449 588550 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 89 450 588551 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 94 451 588552 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG 93 452 588553 2597 2616 Exon 18 CTGTTTTAATTCAATCCCAC 96 453 588554 2598 2617 Exon 18 GCTGTTTTAATTCAATCCCA 98 454 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 97 239 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 95 239 588555 2600 2619 Exon 18 CAGCTGTTTTAATTCAATCC 93 455 588556 2601 2620 Exon 18 GCAGCTGTTTTAATTCAATC 96 456 588557 2602 2621 Exon 18 CGCAGCTGTTTTAATTCAAT 98 457 588558 2603 2622 Exon 18 TCGCAGCTGTTTTAATTCAA 95 458 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 97 317 588559 2605 2624 Exon 18 TGTCGCAGCTGTTTTAATTC 95 459 588560 2606 2625 Exon 18 TTGTCGCAGCTGTTTTAATT 92 460 588561 2607 2626 Exon 18 GTTGTCGCAGCTGTTTTAAT 93 461 532952 2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 88 395 588562 2609 2628 Exon 18/ TTGTTGTCGCAGCTGTTTTA 90 462 Repeat 588563 2610 2629 Exon 18/ TTTGTTGTCGCAGCTGTTTT 89 463 Repeat 588564 2611 2630 Exon 18/ TTTTGTTGTCGCAGCTGTTT 92 464 Repeat 588565 2612 2631 Exon 18/ TTTTTGTTGTCGCAGCTGTT 88 465 Repeat

TABLE 131 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ ID ID SEQ ID NO: 1 NO: 1 ID NO NO: 2 SEQ ISIS start stop Target % 2: start stop ID NO site site region Sequence inhibition site site NO: 588685 n/a n/a Exon 1 GGATCCAGCTCACTCCCCTG 48 1596 1615 466 588686 n/a n/a Exon 1 AAATAAGGATCCAGCTCACT 29 1602 n/a 467 588688 n/a n/a Exon 1 GACCAGAAATAAGGATCCAG 58 1608 1627 468 588690 n/a n/a Exon 1 CTTAGGGACCAGAAATAAGG 45 1614 1633 469 588692 n/a n/a Exon 1 CACCCACTTAGGGACCAGAA 36 1620 1639 470 588694 n/a n/a Exon 1 ACCACCCACTTAGGGACCAG 47 1622 1641 471 588696 n/a n/a Exon 1 AGGTCCAGGACTCTCCCCTT 96 1685 1704 472 588698 n/a n/a Exon 1 AAGGTCCAGGACTCTCCCCT 96 1686 1705 473 588700 n/a n/a Exon 1 AAACTGCAGAAGTCCCACCC 2 1716 1735 474 588586 30 49 Exon 1 GGAGGGCCCCGCTGAGCTGC 59 1751 1770 475 588587 48 67 Exon 1 TCCCGGAACATCCAAGCGGG 45 1769 1788 476 588588 56 75 Exon 1 CATCACTTTCCCGGAACATC 39 1777 n/a 477 588589 151 170 Exon 1 CTGGTCACATTCCCTTCCCC 29 1872 1891 478 588590 157 176 Exon 1 CTAGACCTGGTCACATTCCC 47 1878 1897 479 588591 339 358 Exon 1-2 GGAGTGGTGGTCACACCTCC 44 n/a n/a 480 Junction 588592 384 403 Exon 2 ACCCCCTCCAGAGAGCAGGA 43 2192 2211 481 588593 390 409 Exon 2 ATCTCTACCCCCTCCAGAGA 34 2198 2217 482 588594 467 486 Exon 2 GGTACGGGTAGAAGCCAGAA 17 2275 2294 483 588595 671 690 Exon 3 GGAGAGTGTAACCGTCATAG 37 2879 2898 484 588596 689 708 Exon 3 TGCGATTGGCAGAGCCCCGG 18 2897 2916 485 588597 695 714 Exon 3 GGCAGGTGCGATTGGCAGAG 32 2903 2922 486 588598 707 726 Exon 3 GGCCATTCACTTGGCAGGTG 45 2915 2934 487 588599 738 757 Exon 3 TTGTCACAGATCGCTGTCTG 52 2946 2965 488 588600 924 943 Exon 4-5 AAGGAGTCTTGGCAGGAAGG 39 n/a n/a 489 Junction 588601 931 950 Exon 4-5 GTACATGAAGGAGTCTTGGC 37 n/a n/a 490 Junction 588602 959 978 Exon 5 AAGCTTCGGCCACCTCTTGA 21 3542 3561 491 588603 1089 1108 Exon 6 CCATCTAGCACCAGGTAGAT 22 3773 3792 492 588604 1108 1127 Exon 6 GGCCCCAATGCTGTCTGATC 21 3792 3811 493 588606 1150 1169 Exon 6 AATTAAGTTGACTAGACACT 56 3834 3853 494 588608 1162 1181 Exon 6-7 TGCCACCTTCTCAATTAAGT 50   19 495 Junction 588578 1167 1186 Exon 6-7 TAACTTGCCACCTTCTCAAT 23 n/a n/a 496 Junction 588579 1169 1188 Exon 6-7 CATAACTTGCCACCTTCTCA 23 n/a n/a 497 Junction 532692 1171 1190 Exon 6-7 ACCATAACTTGCCACCTTCT 15 n/a n/a 90 Junction 588580 1173 1192 Exon 6-7 ACACCATAACTTGCCACCTT 16 n/a n/a 498 Junction 588581 1175 1194 Exon 6-7 TCACACCATAACTTGCCACC 14 4151 4170 499 Junction 588610 1319 1338 Exon 8 TAGTCCCTGACTTCAACTTG 50 4612 4631 500 588612 1325 1344 Exon 8 TGGTGTTAGTCCCTGACTTC 47 4618 4637 501 588614 1396 1415 Exon 8 GCGGTTCCAGCCTTCAGGAG 47 4689 4708 502 588616 1421 1440 Exon 8 TCATGAGGATGATGACATGG 51 4714 4733 503 588618 1446 1465 Exon 9 CCGCCCATGTTGTGCAATCC 18 5020 5039 504 588620 1458 1477 Exon 9 GTAATTGGGTCCCCGCCCAT 40 5032 5051 505 588623 1482 1501 Exon 9 AAGTCCCGGATCTCATCAAT 40 5056 5075 506 588624 1542 1561 Exon 9- AACACATAGACATCCAGATA 45 n/a n/a 507 10 Junction 588626 1585 1604 Exon 10 CAAAGCATTGATGTTCACTT 43 5234 5253 508 588628 1621 1640 Exon 10 TTTGAACACATGTTGCTCAT 45 5270 5289 509 588631 1646 1665 Exon 10 CTTCCAGGTTTTCCATATCC 53 5295 5314 510 588632 1647 1666 Exon 10 TCTTCCAGGTTTTCCATATC 56 5296 5315 511 588634 1689 1708 Exon 11 AGACTCAGAGACTGGCTTTC 35 5830 5849 512 588636 1749 1768 Exon 11 GCCTGCCATGGTTGCTTGTG 55 5890 5909 513 588638 1763 1782 Exon 11 TGACTGAGATCTTGGCCTGC 78 5904 5923 514 588640 1912 1931 Exon 13 TTCTATCTCCAGGTCCCGCT 95 6406 6425 515 588642 1982 2001 Exon 13 AGTCATAAAATTCAGGAATT 44 6476 6495 516 588645 2073 2092 Exon 14 CGAGTTGTTCCCTCGGTGCA 40 6662 6681 517 588646 2085 2104 Exon 14 AGCCTCAAAGCTCGAGTTGT 57 6674 6693 518 588648 2091 2110 Exon 14 GGAGGAAGCCTCAAAGCTCG 48 6680 6699 519 588651 2097 2116 Exon 14 GTAGTTGGAGGAAGCCTCAA 40 6686 6705 520 588652 2103 2122 Exon 14 CAAGTGGTAGTTGGAGGAAG 43 6692 6711 521 588654 2166 2185 Exon 15 TCCTCAGACACAAACAGAGC 13 6954 6973 522 588656 2172 2191 Exon 15 TTCTCCTCCTCAGACACAAA 55 6960 6979 523 588658 2196 2215 Exon 15 TAGACCTCCTTCCGAGTCAG 44 6984 7003 524 588660 2202 2221 Exon 15 TTGATGTAGACCTCCTTCCG 50 6990 7009 525 588582 2219 2238 Exon 15- CTTTCTTATCCCCATTCTTG 19 n/a n/a 526 16 Junction 588583 2221 2240 Exon 15- GCCTTTCTTATCCCCATTCT 14 n/a n/a 527 16 Junction 532775 2223 2242 Exon 15- CTGCCTTTCTTATCCCCATT 3 n/a n/a 203 16 Junction 588584 2225 2244 Exon 15- AGCTGCCTTTCTTATCCCCA 18 n/a n/a 528 16 Junction 588662 2226 2245 Exon 15- CAGCTGCCTTTCTTATCCCC 27 n/a n/a 529 16 Junction 588585 2227 2246 Exon 15- ACAGCTGCCTTTCTTATCCC 59 n/a n/a 530 16 Junction 588664 2238 2257 Exon 16 GCATCTCTCTCACAGCTGCC 49 7122 7141 531 588666 2276 2295 Exon 16 AGATGTCCTTGACTTTGTCA 41 7160 7179 532 588668 2330 2349 Exon 16 CAGCATAGGGACTCACTCCT 41 7214 7233 533 588670 2361 2380 Exon 16- CCGCCAGAATCACCTCTGCA 43 n/a n/a 534 17 Junction 588672 2397 2416 Exon 17 TGAATGAAACGACTTCTCTT 52 7362 7381 535 588674 2430 2449 Exon 18 ACATCCACTACTCCCCAGCT 39 7665 7684 536 588676 2448 2467 Exon 18 CGCTTCTGGTTTTTGCAGAC 69 7683 7702 537 588678 2454 2473 Exon 18 TTTTGCCGCTTCTGGTTTTT 46 7689 7708 538 588680 2466 2485 Exon 18 GCAGGTACCTGCTTTTGCCG 47 7701 7720 539 588682 2532 2551 Exon 18 TCTTGGAGTTTCTCCTTCAG 58 7767 7786 540 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 10 7834 7853 239 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 11 7839 7858 317

Example 118: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 3,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 4-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE, 3-10-4 MOE, 3-10-7 MOE, 6-7-6-MOE, 6-8-6 MOE, or 5-7-5 MOE gapmers, or as deoxy, MOE, and (S)-cEt oligonucleotides.

The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four and five nucleosides respectively. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-7-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and four nucleosides respectively. The 3-10-7 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and seven nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 132 Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Start stop Target % Start Stop SEQ ISIS NO site site region Sequence inhibition site site Motif ID NO: 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 10 7834 7853 eeeeeddddddddddeeeee 239 588884 48 63 Exon 1 GGAACATCCAAGCGGG 79 1769 1784 eekddddddddddkke 541 588872 154 169 Exon 1 TGGTCACATTCCCTTC 91 1875 1890 eekddddddddddkke 542 588873 156 171 Exon 1 CCTGGTCACATTCCCT 91 1877 1892 eekddddddddddkke 543 588874 158 173 Exon 1 GACCTGGTCACATTCC 91 1879 1894 eekddddddddddkke 544 588878 1171 1186 Exon 6-7 TAACTTGCCACCTTCT 92 n/a n/a eekddddddddddkke 545 Junction 588879 1173 1188 Exon 6-7 CATAACTTGCCACCTT 94 n/a n/a eekddddddddddkke 546 Junction 588880 1175 1190 Exon 6-7 ACCATAACTTGCCACC 89 4151 4166 eekddddddddddkke 547 Junction 588869 2193 2208 Exon 15 CCTTCCGAGTCAGCTT 17 6981 6996 eekddddddddddkke 548 588870 2195 2210 Exon 15 CTCCTTCCGAGTCAGC 78 6983 6998 eekddddddddddkke 549 588871 2197 2212 Exon 15 ACCTCCTTCCGAGTCA 80 6985 7000 eekddddddddddkke 550 588881 2223 2238 Exon 15- CTTTCTTATCCCCATT 93 n/a n/a eekddddddddddkke 551 16 Junction 588882 2225 2240 Exon 15- GCCTTTCTTATCCCCA 88 n/a n/a eekddddddddddkke 552 16 Junction 588883 2227 2242 Exon 15- CTGCCTTTCTTATCCC 90 n/a n/a eekddddddddddkke 553 16 Junction 588875 2457 2472 Exon 18 TTTGCCGCTTCTGGTT 81 7692 7707 eekddddddddddkke 554 588876 2459 2474 Exon 18 CTTTTGCCGCTTCTGG 95 7694 7709 eekddddddddddkke 555 588877 2461 2476 Exon 18 TGCTTTTGCCGCTTCT 91 7696 7711 eekddddddddddkke 556 588807 2551 2566 Exon 18 AAACCCAAATCCTCAT 82 7786 7801 eekddddddddddkke 557 588808 2553 2568 Exon 18 GAAAACCCAAATCCTC 69 7788 7803 eekddddddddddkke 558 588809 2555 2570 Exon 18 TAGAAAACCCAAATCC 51 7790 7805 eekddddddddddkke 559 588810 2556 2571 Exon 18 ATAGAAAACCCAAATC 23 7791 7806 eekddddddddddkke 560 588811 2559 2574 Exon 18 CTTATAGAAAACCCAA 13 7794 7809 eekddddddddddkke 561 588812 2560 2575 Exon 18 CCTTATAGAAAACCCA 29 7795 7810 eekddddddddddkke 562 588813 2561 2576 Exon 18 CCCTTATAGAAAACCC 53 7796 7811 eekddddddddddkke 563 588814 2562 2577 Exon 18 CCCCTTATAGAAAACC 86 7797 7812 eekddddddddddkke 564 588815 2563 2578 Exon 18 ACCCCTTATAGAAAAC 76 7798 7813 eekddddddddddkke 565 588816 2564 2579 Exon 18 AACCCCTTATAGAAAA 33 7799 7814 eekddddddddddkke 566 588817 2565 2580 Exon 18 AAACCCCTTATAGAAA 48 7800 7815 eekddddddddddkke 567 588818 2566 2581 Exon 18 GAAACCCCTTATAGAA 44 7801 7816 eekddddddddddkke 568 588819 2567 2582 Exon 18 GGAAACCCCTTATAGA 74 7802 7817 eekddddddddddkke 569 588820 2568 2583 Exon 18 AGGAAACCCCTTATAG 68 7803 7818 eekddddddddddkke 570 588821 2569 2584 Exon 18 CAGGAAACCCCTTATA 45 7804 7819 eekddddddddddkke 571 588822 2570 2585 Exon 18 GCAGGAAACCCCTTAT 50 7805 7820 eekddddddddddkke 572 588823 2571 2586 Exon 18 AGCAGGAAACCCCTTA 54 7806 7821 eekddddddddddkke 573 588824 2572 2587 Exon 18 CAGCAGGAAACCCCTT 35 7807 7822 eekddddddddddkke 574 588825 2573 2588 Exon 18 CCAGCAGGAAACCCCT 11 7808 7823 eekddddddddddkke 575 588826 2574 2589 Exon 18 TCCAGCAGGAAACCCC 19 7809 7824 eekddddddddddkke 576 588827 2575 2590 Exon 18 GTCCAGCAGGAAACCC 42 7810 7825 eekddddddddddkke 577 588828 2576 2591 Exon 18 TGTCCAGCAGGAAACC 0 7811 7826 eekddddddddddkke 578 588829 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 49 7812 7827 eekddddddddddkke 579 588830 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 11 7813 7828 eekddddddddddkke 580 588831 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 20 7814 7829 eekddddddddddkke 581 588832 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 19 7815 7830 eekddddddddddkke 582 588833 2581 2596 Exon 18 GCCCCTGTCCAGCAGG 12 7816 7831 eekddddddddddkke 583 588834 2582 2597 Exon 18 CGCCCCTGTCCAGCAG 10 7817 7832 eekddddddddddkke 584 588835 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 13 7818 7833 eekddddddddddkke 585 588836 2584 2599 Exon 18 CACGCCCCTGTCCAGC 13 7819 7834 eekddddddddddkke 586 588837 2585 2600 Exon 18 CCACGCCCCTGTCCAG 39 7820 7835 eekddddddddddkke 587 588838 2586 2601 Exon 18 CCCACGCCCCTGTCCA 54 7821 7836 eekddddddddddkke 588 588839 2587 2602 Exon 18 TCCCACGCCCCTGTCC 51 7822 7837 eekddddddddddkke 589 588840 2588 2603 Exon 18 ATCCCACGCCCCTGTC 65 7823 7838 eekddddddddddkke 590 588841 2589 2604 Exon 18 AATCCCACGCCCCTGT 59 7824 7839 eekddddddddddkke 591 588842 2590 2605 Exon 18 CAATCCCACGCCCCTG 70 7825 7840 eekddddddddddkke 592 588843 2591 2606 Exon 18 TCAATCCCACGCCCCT 0 7826 7841 eekddddddddddkke 593 588844 2592 2607 Exon 18 TTCAATCCCACGCCCC 48 7827 7842 eekddddddddddkke 594 588845 2593 2608 Exon 18 ATTCAATCCCACGCCC 46 7828 7843 eekddddddddddkke 595 588846 2594 2609 Exon 18 AATTCAATCCCACGCC 67 7829 7844 eekddddddddddkke 596 588847 2595 2610 Exon 18 TAATTCAATCCCACGC 75 7830 7845 eekddddddddddkke 597 588848 2596 2611 Exon 18 TTAATTCAATCCCACG 76 7831 7846 eekddddddddddkke 598 588849 2597 2612 Exon 18 TTTAATTCAATCCCAC 94 7832 7847 eekddddddddddkke 599 588850 2598 2613 Exon 18 TTTTAATTCAATCCCA 91 7833 7848 eekddddddddddkke 600 588851 2599 2614 Exon 18 GTTTTAATTCAATCCC 91 7834 7849 eekddddddddddkke 601 588852 2600 2615 Exon 18 TGTTTTAATTCAATCC 78 7835 7850 eekddddddddddkke 602 588853 2601 2616 Exon 18 CTGTTTTAATTCAATC 81 7836 7851 eekddddddddddkke 603 588854 2602 2617 Exon 18 GCTGTTTTAATTCAAT 63 7837 7852 eekddddddddddkke 604 588855 2603 2618 Exon 18 AGCTGTTTTAATTCAA 65 7838 7853 eekddddddddddkke 605 588856 2604 2619 Exon 18 CAGCTGTTTTAATTCA 76 7839 7854 eekddddddddddkke 606 588857 2605 2620 Exon 18 GCAGCTGTTTTAATTC 89 7840 7855 eekddddddddddkke 607 588858 2606 2621 Exon 18 CGCAGCTGTTTTAATT 89 7841 7856 eekddddddddddkke 608 588859 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 89 7842 7857 eekddddddddddkke 609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 76 7843 7858 eekddddddddddkke 610 588861 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 87 7844 7859 eekddddddddddkke 611 588862 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 85 7845 7860 eekddddddddddkke 612 588863 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 87 7846 7861 eekddddddddddkke 613 588864 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 67 7847 7862 eekddddddddddkke 614 588865 2613 2628 Exon 18 TTGTTGTCGCAGCTGT 51 n/a n/a eekddddddddddkke 615 588866 2614 2629 Exon 18 TTTGTTGTCGCAGCTG 95 n/a n/a eekddddddddddkke 616 588867 2615 2630 Exon 18 TTTTGTTGTCGCAGCT 92 n/a n/a eekddddddddddkke 617 588868 2616 2631 Exon 18 TTTTTGTTGTCGCAGC 66 n/a n/a eekddddddddddkke 618

TABLE 133 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ ID ID SEQ SEQ NO: 1 NO: 1 ID NO: ID NO: SEQ ISIS start stop Target % 2 start 2 stop ID NO site site region Sequence inhibition site site NO: 588685 n/a n/a Exon 1 GGATCCAGCTCACTCCCCTG 14 1596 1615 466 588686 n/a n/a Exon 1 AAATAAGGATCCAGCTCACT 2 1602 1621 467 588688 n/a n/a Exon 1 GACCAGAAATAAGGATCCAG 3 1608 1627 468 588690 n/a n/a Exon 1 CTTAGGGACCAGAAATAAGG 10 1614 1633 469 588692 n/a n/a Exon 1 CACCCACTTAGGGACCAGAA 23 1620 1639 470 588694 n/a n/a Exon 1 ACCACCCACTTAGGGACCAG 23 1622 1641 471 588696 n/a n/a Exon 1 AGGTCCAGGACTCTCCCCTT 15 1685 1704 472 588698 n/a n/a Exon 1 AAGGTCCAGGACTCTCCCCT 19 1686 1705 473 588700 n/a n/a Exon 1 AAACTGCAGAAGTCCCACCC 16 1716 1735 474 588586 30 49 Exon 1 GGAGGGCCCCGCTGAGCTGC 11 1751 1770 475 588587 48 67 Exon 1 TCCCGGAACATCCAAGCGGG 14 1769 1788 476 588588 56 75 Exon 1 CATCACTTTCCCGGAACATC 18 1777 1796 477 588589 151 170 Exon 1 CTGGTCACATTCCCTTCCCC 59 1872 1891 478 588590 157 176 Exon 1 CTAGACCTGGTCACATTCCC 59 1878 1897 479 588591 339 358 Exon 1-2 GGAGTGGTGGTCACACCTCC 45 n/a n/a 480 Junction 588592 384 403 Exon 2 ACCCCCTCCAGAGAGCAGGA 39 2192 2211 481 588593 390 409 Exon 2 ATCTCTACCCCCTCCAGAGA 29 2198 2217 482 588594 467 486 Exon 2 GGTACGGGTAGAAGCCAGAA 47 2275 2294 483 588595 671 690 Exon 3 GGAGAGTGTAACCGTCATAG 44 2879 2898 484 588596 689 708 Exon 3 TGCGATTGGCAGAGCCCCGG 43 2897 2916 638 588597 695 714 Exon 3 GGCAGGTGCGATTGGCAGAG 34 2903 2922 486 588598 707 726 Exon 3 GGCCATTCACTTGGCAGGTG 17 2915 2934 487 588599 738 757 Exon 3 TTGTCACAGATCGCTGTCTG 37 2946 2965 488 588600 924 943 Exon 3-4 AAGGAGTCTTGGCAGGAAGG 18 n/a n/a 489 Junction 588601 931 950 Exon 3-4 GTACATGAAGGAGTCTTGGC 32 n/a n/a 490 Junction 588602 959 978 Exon 5 AAGCTTCGGCCACCTCTTGA 45 3542 3561 491 588603 1089 1108 Exon 6 CCATCTAGCACCAGGTAGAT 52 3773 3792 492 588604 1108 1127 Exon 6 GGCCCCAATGCTGTCTGATC 39 3792 3811 493 588606 1150 1169 Exon 6 AATTAAGTTGACTAGACACT 37 3834 3853 494 588608 1162 1181 Exon 6-7 TGCCACCTTCTCAATTAAGT 21 n/a n/a 648 Junction 588578 1167 1186 Exon 6-7 TAACTTGCCACCTTCTCAAT 22 n/a n/a 496 Junction 588579 1169 1188 Exon 6-7 CATAACTTGCCACCTTCTCA 21 n/a n/a 497 Junction 532692 1171 1190 Exon 6-7 ACCATAACTTGCCACCTTCT 56 n/a n/a 90 Junction 588580 1173 1192 Exon 6-7 ACACCATAACTTGCCACCTT 50 n/a n/a 498 Junction 588581 1175 1194 Exon 7 TCACACCATAACTTGCCACC 50 4151 4170 499 588610 1319 1338 Exon 8 TAGTCCCTGACTTCAACTTG 47 4612 4631 500 588612 1325 1344 Exon 8 TGGTGTTAGTCCCTGACTTC 47 4618 4637 501 588614 1396 1415 Exon 8 GCGGTTCCAGCCTTCAGGAG 51 4689 4708 502 588616 1421 1440 Exon 8 TCATGAGGATGATGACATGG 18 4714 4733 503 588618 1446 1465 Exon 9 CCGCCCATGTTGTGCAATCC 40 5020 5039 504 588620 1458 1477 Exon 9 GTAATTGGGTCCCCGCCCAT 40 5032 5051 505 588623 1482 1501 Exon 9 AAGTCCCGGATCTCATCAAT 45 5056 5075 506 588624 1542 1561 Exon 9-10 AACACATAGACATCCAGATA 43 n/a n/a 507 Junction 588626 1585 1604 Exon 10 CAAAGCATTGATGTTCACTT 45 5234 5253 508 588628 1621 1640 Exon 10 TTTGAACACATGTTGCTCAT 53 5270 5289 509 588631 1646 1665 Exon 10 CTTCCAGGTTTTCCATATCC 56 5295 5314 510 588632 1647 1666 Exon 10 TCTTCCAGGTTTTCCATATC 35 5296 5315 511 588634 1689 1708 Exon 11 AGACTCAGAGACTGGCTTTC 55 5830 5849 512 588636 1749 1768 Exon 11 GCCTGCCATGGTTGCTTGTG 78 5890 5909 513 588638 1763 1782 Exon 11 TGACTGAGATCTTGGCCTGC 95 5904 5923 514 588640 1912 1931 Exon 13 TTCTATCTCCAGGTCCCGCT 44 6406 6425 515 588642 1982 2001 Exon 13 AGTCATAAAATTCAGGAATT 40 6476 6495 516 588645 2073 2092 Exon 14 CGAGTTGTTCCCTCGGTGCA 57 6662 6681 517 588646 2085 2104 Exon 14 AGCCTCAAAGCTCGAGTTGT 48 6674 6693 518 588648 2091 2110 Exon 14 GGAGGAAGCCTCAAAGCTCG 40 6680 6699 519 588651 2097 2116 Exon 14 GTAGTTGGAGGAAGCCTCAA 43 6686 6705 520 588652 2103 2122 Exon 14 CAAGTGGTAGTTGGAGGAAG 13 6692 6711 521 588654 2166 2185 Exon 15 TCCTCAGACACAAACAGAGC 55 6954 6973 522 588656 2172 2191 Exon 15 TTCTCCTCCTCAGACACAAA 44 6960 6979 523 588658 2196 2215 Exon 15 TAGACCTCCTTCCGAGTCAG 50 6984 7003 524 588660 2202 2221 Exon 15 TTGATGTAGACCTCCTTCCG 27 6990 7009 525 588582 2219 2238 Exon 15- CTTTCTTATCCCCATTCTTG 49 n/a n/a 526 16 Junction 588583 2221 2240 Exon 15- GCCTTTCTTATCCCCATTCT 41 n/a n/a 527 16 Junction 532775 2223 2242 Exon 15- CTGCCTTTCTTATCCCCATT 41 n/a n/a 203 16 Junction 588584 2225 2244 Exon 15- AGCTGCCTTTCTTATCCCCA 43 n/a n/a 528 16 Junction 588662 2226 2245 Exon 15- CAGCTGCCTTTCTTATCCCC 52 n/a n/a 529 16 Junction 588585 2227 2246 Exon 15- ACAGCTGCCTTTCTTATCCC 39 n/a n/a 530 16 Junction 588664 2238 2257 Exon 16 GCATCTCTCTCACAGCTGCC 69 7122 7141 531 588666 2276 2295 Exon 16 AGATGTCCTTGACTTTGTCA 46 7160 7179 532 588668 2330 2349 Exon 16 CAGCATAGGGACTCACTCCT 47 7214 7233 533 588670 2361 2380 Exon 16- CCGCCAGAATCACCTCTGCA 58 n/a n/a 534 17 Junction 588672 2397 2416 Exon 17 TGAATGAAACGACTTCTCTT 48 7362 7381 535 588674 2430 2449 Exon 18 ACATCCACTACTCCCCAGCT 29 7665 7684 536 588676 2448 2467 Exon 18 CGCTTCTGGTTTTTGCAGAC 58 7683 7702 537 588678 2454 2473 Exon 18 TTTTGCCGCTTCTGGTTTTT 45 7689 7708 538 588680 2466 2485 Exon 18 GCAGGTACCTGCTTTTGCCG 36 7701 7720 539 588682 2532 2551 Exon 18 TCTTGGAGTTTCTCCTTCAG 47 7767 7786 540 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 96 7834 7853 239 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 96 7839 7858 317

TABLE 134 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site Motif NO: 598973 2552 2568 Exon 18 GAAAACCCAAATCCTCA 40 7787 7803 3-10-4 619 599036 2552 2568 Exon 18 GAAAACCCAAATCCTCA 18 7787 7803 5-7-5 619 598974 2553 2569 Exon 18 AGAAAACCCAAATCCTC 28 7788 7804 3-10-4 620 599037 2553 2569 Exon 18 AGAAAACCCAAATCCTC 19 7788 7804 5-7-5 620 598975 2554 2570 Exon 18 TAGAAAACCCAAATCCT 15 7789 7805 3-10-4 621 599038 2554 2570 Exon 18 TAGAAAACCCAAATCCT 32 7789 7805 5-7-5 621 598976 2555 2571 Exon 18 ATAGAAAACCCAAATCC 12 7790 7806 3-10-4 622 599039 2555 2571 Exon 18 ATAGAAAACCCAAATCC 7 7790 7806 5-7-5 622 598977 2557 2573 Exon 18 TTATAGAAAACCCAAAT 13 7792 7808 3-10-4 623 599040 2557 2573 Exon 18 TTATAGAAAACCCAAAT 13 7792 7808 5-7-5 623 598978 2558 2574 Exon 18 CTTATAGAAAACCCAAA 0 7793 7809 3-10-4 624 599041 2558 2574 Exon 18 CTTATAGAAAACCCAAA 0 7793 7809 5-7-5 624 598979 2559 2575 Exon 18 CCTTATAGAAAACCCAA 8 7794 7810 3-10-4 625 599042 2559 2575 Exon 18 CCTTATAGAAAACCCAA 19 7794 7810 5-7-5 625 598980 2560 2576 Exon 18 CCCTTATAGAAAACCCA 42 7795 7811 3-10-4 626 599043 2560 2576 Exon 18 CCCTTATAGAAAACCCA 10 7795 7811 5-7-5 626 598981 2561 2577 Exon 18 CCCCTTATAGAAAACCC 20 7796 7812 3-10-4 627 599044 2561 2577 Exon 18 CCCCTTATAGAAAACCC 12 7796 7812 5-7-5 627 598982 2562 2578 Exon 18 ACCCCTTATAGAAAACC 10 7797 7813 3-10-4 628 599045 2562 2578 Exon 18 ACCCCTTATAGAAAACC 3 7797 7813 5-7-5 628 598983 2563 2579 Exon 18 AACCCCTTATAGAAAAC 0 7798 7814 3-10-4 629 599046 2563 2579 Exon 18 AACCCCTTATAGAAAAC 18 7798 7814 5-7-5 629 598984 2564 2580 Exon 18 AAACCCCTTATAGAAAA 0 7799 7815 3-10-4 630 599047 2564 2580 Exon 18 AAACCCCTTATAGAAAA 7 7799 7815 5-7-5 630 598985 2565 2581 Exon 18 GAAACCCCTTATAGAAA 0 7800 7816 3-10-4 631 599048 2565 2581 Exon 18 GAAACCCCTTATAGAAA 9 7800 7816 5-7-5 631 598986 2566 2582 Exon 18 GGAAACCCCTTATAGAA 0 7801 7817 3-10-4 632 599049 2566 2582 Exon 18 GGAAACCCCTTATAGAA 18 7801 7817 5-7-5 632 598988 2567 2583 Exon 18 AGGAAACCCCTTATAGA 0 7802 7818 3-10-4 633 599050 2567 2583 Exon 18 AGGAAACCCCTTATAGA 8 7802 7818 5-7-5 633 598989 2568 2584 Exon 18 CAGGAAACCCCTTATAG 0 7803 7819 3-10-4 634 598990 2569 2585 Exon 18 GCAGGAAACCCCTTATA 8 7804 7820 3-10-4 635 598991 2570 2586 Exon 18 AGCAGGAAACCCCTTAT 25 7805 7821 3-10-4 636 598992 2571 2587 Exon 18 CAGCAGGAAACCCCTTA 12 7806 7822 3-10-4 637 598993 2572 2588 Exon 18 CCAGCAGGAAACCCCTT 37 7807 7823 3-10-4 638 598994 2573 2589 Exon 18 TCCAGCAGGAAACCCCT 29 7808 7824 3-10-4 639 598995 2574 2590 Exon 18 GTCCAGCAGGAAACCCC 42 7809 7825 3-10-4 640 598996 2575 2591 Exon 18 TGTCCAGCAGGAAACCC 36 7810 7826 3-10-4 641 598997 2576 2592 Exon 18 CTGTCCAGCAGGAAACC 18 7811 7827 3-10-4 642 598998 2577 2593 Exon 18 CCTGTCCAGCAGGAAAC 27 7812 7828 3-10-4 643 598999 2578 2594 Exon 18 CCCTGTCCAGCAGGAAA 61 7813 7829 3-10-4 644 599000 2580 2596 Exon 18 GCCCCTGTCCAGCAGGA 71 7815 7831 3-10-4 645 599001 2581 2597 Exon 18 CGCCCCTGTCCAGCAGG 80 7816 7832 3-10-4 646 599002 2582 2598 Exon 18 ACGCCCCTGTCCAGCAG 68 7817 7833 3-10-4 647 599003 2583 2599 Exon 18 CACGCCCCTGTCCAGCA 71 7818 7834 3-10-4 648 599004 2584 2600 Exon 18 CCACGCCCCTGTCCAGC 76 7819 7835 3-10-4 649 599005 2585 2601 Exon 18 CCCACGCCCCTGTCCAG 70 7820 7836 3-10-4 650 599006 2586 2602 Exon 18 TCCCACGCCCCTGTCCA 65 7821 7837 3-10-4 651 599007 2587 2603 Exon 18 ATCCCACGCCCCTGTCC 60 7822 7838 3-10-4 652 599008 2588 2604 Exon 18 AATCCCACGCCCCTGTC 72 7823 7839 3-10-4 653 599009 2589 2605 Exon 18 CAATCCCACGCCCCTGT 79 7824 7840 3-10-4 654 599010 2590 2606 Exon 18 TCAATCCCACGCCCCTG 73 7825 7841 3-10-4 655 599011 2591 2607 Exon 18 TTCAATCCCACGCCCCT 79 7826 7842 3-10-4 656 599012 2592 2608 Exon 18 ATTCAATCCCACGCCCC 67 7827 7843 3-10-4 657 599013 2593 2609 Exon 18 AATTCAATCCCACGCCC 65 7828 7844 3-10-4 658 599014 2594 2610 Exon 18 TAATTCAATCCCACGCC 74 7829 7845 3-10-4 659 599015 2595 2611 Exon 18 TTAATTCAATCCCACGC 71 7830 7846 3-10-4 660 599016 2596 2612 Exon 18 TTTAATTCAATCCCACG 48 7831 7847 3-10-4 661 599017 2597 2613 Exon 18 TTTTAATTCAATCCCAC 34 7832 7848 3-10-4 662 599018 2598 2614 Exon 18 GTTTTAATTCAATCCCA 56 7833 7849 3-10-4 663 599019 2599 2615 Exon 18 TGTTTTAATTCAATCCC 60 7834 7850 3-10-4 664 599020 2600 2616 Exon 18 CTGTTTTAATTCAATCC 0 7835 7851 3-10-4 665 599021 2601 2617 Exon 18 GCTGTTTTAATTCAATC 33 7836 7852 3-10-4 666 599022 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 17 7837 7853 3-10-4 667 599023 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 52 7838 7854 3-10-4 668 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 86 7839 7858 5-10-5 317 599024 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 88 7839 7855 3-10-4 669 599025 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 85 7840 7856 3-10-4 670 599026 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 69 7841 7857 3-10-4 671 599027 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 77 7842 7858 3-10-4 672 599028 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 73 7843 7859 3-10-4 673 599029 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 78 7844 7860 3-10-4 674 599030 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 75 7845 7861 3-10-4 675 599031 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 77 7846 7862 3-10-4 676 599032 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 79 n/a n/a 3-10-4 677 Repeat 599033 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 80 n/a n/a 3-10-4 678 Repeat 599034 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 78 n/a n/a 3-10-4 679 Repeat 599035 2615 2631 Exon 18/ TTTTTGTTGTCGCAGCT 63 n/a n/a 3-10-4 680 Repeat

TABLE 135 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site Motif NO: 599098 2552 2568 Exon 18 GAAAACCCAAATCCTCA 57 7787 7803 4-8-5 619 599099 2553 2569 Exon 18 AGAAAACCCAAATCCTC 33 7788 7804 4-8-5 620 599100 2554 2570 Exon 18 TAGAAAACCCAAATCCT 32 7789 7805 4-8-5 621 599101 2555 2571 Exon 18 ATAGAAAACCCAAATCC 47 7790 7806 4-8-5 622 599102 2557 2573 Exon 18 TTATAGAAAACCCAAAT 59 7792 7808 4-8-5 623 599103 2558 2574 Exon 18 CTTATAGAAAACCCAAA 10 7793 7809 4-8-5 624 599104 2559 2575 Exon 18 CCTTATAGAAAACCCAA 3 7794 7810 4-8-5 625 599105 2560 2576 Exon 18 CCCTTATAGAAAACCCA 45 7795 7811 4-8-5 626 599106 2561 2577 Exon 18 CCCCTTATAGAAAACCC 49 7796 7812 4-8-5 627 599107 2562 2578 Exon 18 ACCCCTTATAGAAAACC 35 7797 7813 4-8-5 628 599108 2563 2579 Exon 18 AACCCCTTATAGAAAAC 17 7798 7814 4-8-5 629 599109 2564 2580 Exon 18 AAACCCCTTATAGAAAA 36 7799 7815 4-8-5 630 599110 2565 2581 Exon 18 GAAACCCCTTATAGAAA 20 7800 7816 4-8-5 631 599111 2566 2582 Exon 18 GGAAACCCCTTATAGAA 20 7801 7817 4-8-5 632 599112 2567 2583 Exon 18 AGGAAACCCCTTATAGA 15 7802 7818 4-8-5 633 599113 2568 2584 Exon 18 CAGGAAACCCCTTATAG 19 7803 7819 4-8-5 634 599051 2568 2584 Exon 18 CAGGAAACCCCTTATAG 26 7803 7819 5-7-5 634 599114 2569 2585 Exon 18 GCAGGAAACCCCTTATA 18 7804 7820 4-8-5 635 599052 2569 2585 Exon 18 GCAGGAAACCCCTTATA 21 7804 7820 5-7-5 635 599115 2570 2586 Exon 18 AGCAGGAAACCCCTTAT 31 7805 7821 4-8-5 636 599053 2570 2586 Exon 18 AGCAGGAAACCCCTTAT 25 7805 7821 5-7-5 636 599116 2571 2587 Exon 18 CAGCAGGAAACCCCTTA 39 7806 7822 4-8-5 637 599054 2571 2587 Exon 18 CAGCAGGAAACCCCTTA 36 7806 7822 5-7-5 637 599117 2572 2588 Exon 18 CCAGCAGGAAACCCCTT 46 7807 7823 4-8-5 638 599055 2572 2588 Exon 18 CCAGCAGGAAACCCCTT 22 7807 7823 5-7-5 638 599118 2573 2589 Exon 18 TCCAGCAGGAAACCCCT 40 7808 7824 4-8-5 639 599056 2573 2589 Exon 18 TCCAGCAGGAAACCCCT 32 7808 7824 5-7-5 639 599119 2574 2590 Exon 18 GTCCAGCAGGAAACCCC 50 7809 7825 4-8-5 640 599057 2574 2590 Exon 18 GTCCAGCAGGAAACCCC 46 7809 7825 5-7-5 640 599120 2575 2591 Exon 18 TGTCCAGCAGGAAACCC 30 7810 7826 4-8-5 641 599058 2575 2591 Exon 18 TGTCCAGCAGGAAACCC 52 7810 7826 5-7-5 641 599121 2576 2592 Exon 18 CTGTCCAGCAGGAAACC 31 7811 7827 4-8-5 642 599059 2576 2592 Exon 18 CTGTCCAGCAGGAAACC 24 7811 7827 5-7-5 642 599122 2577 2593 Exon 18 CCTGTCCAGCAGGAAAC 23 7812 7828 4-8-5 643 599060 2577 2593 Exon 18 CCTGTCCAGCAGGAAAC 37 7812 7828 5-7-5 643 599123 2578 2594 Exon 18 CCCTGTCCAGCAGGAAA 51 7813 7829 4-8-5 644 599061 2578 2594 Exon 18 CCCTGTCCAGCAGGAAA 34 7813 7829 5-7-5 644 599124 2580 2596 Exon 18 GCCCCTGTCCAGCAGGA 56 7815 7831 4-8-5 645 599062 2580 2596 Exon 18 GCCCCTGTCCAGCAGGA 51 7815 7831 5-7-5 645 599125 2581 2597 Exon 18 CGCCCCTGTCCAGCAGG 70 7816 7832 4-8-5 646 599063 2581 2597 Exon 18 CGCCCCTGTCCAGCAGG 56 7816 7832 5-7-5 646 599126 2582 2598 Exon 18 ACGCCCCTGTCCAGCAG 76 7817 7833 4-8-5 647 599064 2582 2598 Exon 18 ACGCCCCTGTCCAGCAG 61 7817 7833 5-7-5 647 599127 2583 2599 Exon 18 CACGCCCCTGTCCAGCA 67 7818 7834 4-8-5 648 599065 2583 2599 Exon 18 CACGCCCCTGTCCAGCA 64 7818 7834 5-7-5 648 599066 2584 2600 Exon 18 CCACGCCCCTGTCCAGC 40 7819 7835 5-7-5 649 599067 2585 2601 Exon 18 CCCACGCCCCTGTCCAG 37 7820 7836 5-7-5 650 599068 2586 2602 Exon 18 TCCCACGCCCCTGTCCA 31 7821 7837 5-7-5 651 599069 2587 2603 Exon 18 ATCCCACGCCCCTGTCC 39 7822 7838 5-7-5 652 599070 2588 2604 Exon 18 AATCCCACGCCCCTGTC 59 7823 7839 5-7-5 653 599071 2589 2605 Exon 18 CAATCCCACGCCCCTGT 63 7824 7840 5-7-5 657 599072 2590 2606 Exon 18 TCAATCCCACGCCCCTG 74 7825 7841 5-7-5 655 599073 2591 2607 Exon 18 TTCAATCCCACGCCCCT 53 7826 7842 5-7-5 656 599074 2592 2608 Exon 18 ATTCAATCCCACGCCCC 56 7827 7843 5-7-5 657 599075 2593 2609 Exon 18 AATTCAATCCCACGCCC 49 7828 7844 5-7-5 658 599076 2594 2610 Exon 18 TAATTCAATCCCACGCC 54 7829 7845 5-7-5 659 599077 2595 2611 Exon 18 TTAATTCAATCCCACGC 79 7830 7846 5-7-5 660 599078 2596 2612 Exon 18 TTTAATTCAATCCCACG 67 7831 7847 5-7-5 661 599079 2597 2613 Exon 18 TTTTAATTCAATCCCAC 69 7832 7848 5-7-5 662 599080 2598 2614 Exon 18 GTTTTAATTCAATCCCA 79 7833 7849 5-7-5 663 599081 2599 2615 Exon 18 TGTTTTAATTCAATCCC 57 7834 7850 5-7-5 664 599082 2600 2616 Exon 18 CTGTTTTAATTCAATCC 50 7835 7851 5-7-5 665 599083 2601 2617 Exon 18 GCTGTTTTAATTCAATC 67 7836 7852 5-7-5 666 599084 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 60 7837 7853 5-7-5 667 599085 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 71 7838 7854 5-7-5 668 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 82 7839 7858 5-10-5 317 599086 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 81 7839 7855 5-7-5 669 599087 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 88 7840 7856 5-7-5 670 599088 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 84 7841 7857 5-7-5 671 599089 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 81 7842 7858 5-7-5 672 599090 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 77 7843 7859 5-7-5 673 599091 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 74 7844 7860 5-7-5 674 599092 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 66 7845 7861 5-7-5 675 599093 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 89 7846 7862 5-7-5 676 599094 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 82 n/a n/a 5-7-5 677 Repeat 599095 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 87 n/a n/a 5-7-5 678 Repeat 599096 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 85 n/a n/a 5-7-5 679 Repeat 599097 2615 2631 Exon 18/ TTTTTGTTGTCGCAGCT 78 n/a n/a 5-7-5 680 Repeat

TABLE 136 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 ID NO: NO: 2 SEQ ISIS start stop Target % 2 start stop ID NO site site region Sequence inhibition site site Motif NO: 599510 2552 2570 Exon 18 TAGAAAACCCAAATCCTCA 45 7787 7805 5-9-5 681 599331 2553 2571 Exon 18 ATAGAAAACCCAAATCCTC 46 7788 7806 5-9-5 682 599332 2554 2572 Exon 18 TATAGAAAACCCAAATCCT 38 7789 7807 5-9-5 683 599333 2556 2574 Exon 18 CTTATAGAAAACCCAAATC 1 7791 7809 5-9-5 684 599334 2557 2575 Exon 18 CCTTATAGAAAACCCAAAT 5 7792 7810 5-9-5 685 599335 2558 2576 Exon 18 CCCTTATAGAAAACCCAAA 34 7793 7811 5-9-5 686 599336 2559 2577 Exon 18 CCCCTTATAGAAAACCCAA 40 7794 7812 5-9-5 687 599337 2560 2578 Exon 18 ACCCCTTATAGAAAACCCA 39 7795 7813 5-9-5 688 599338 2561 2579 Exon 18 AACCCCTTATAGAAAACCC 57 7796 7814 5-9-5 689 599339 2562 2580 Exon 18 AAACCCCTTATAGAAAACC 26 7797 7815 5-9-5 690 599281 2562 2580 Exon 18 AAACCCCTTATAGAAAACC 15 7797 7815 6-7-6 690 599340 2563 2581 Exon 18 GAAACCCCTTATAGAAAAC 17 7798 7816 5-9-5 691 599282 2563 2581 Exon 18 GAAACCCCTTATAGAAAAC 12 7798 7816 6-7-6 691 599341 2564 2582 Exon 18 GGAAACCCCTTATAGAAAA 23 7799 7817 5-9-5 692 599283 2564 2582 Exon 18 GGAAACCCCTTATAGAAAA 18 7799 7817 6-7-6 692 599342 2565 2583 Exon 18 AGGAAACCCCTTATAGAAA 10 7800 7818 5-9-5 693 599284 2565 2583 Exon 18 AGGAAACCCCTTATAGAAA 14 7800 7818 6-7-6 693 599343 2566 2584 Exon 18 CAGGAAACCCCTTATAGAA 10 7801 7819 5-9-5 694 599285 2566 2584 Exon 18 CAGGAAACCCCTTATAGAA 13 7801 7819 6-7-6 694 599344 2567 2585 Exon 18 GCAGGAAACCCCTTATAGA 22 7802 7820 5-9-5 695 599286 2567 2585 Exon 18 GCAGGAAACCCCTTATAGA 31 7802 7820 6-7-6 695 599345 2568 2586 Exon 18 AGCAGGAAACCCCTTATAG 19 7803 7821 5-9-5 696 599287 2568 2586 Exon 18 AGCAGGAAACCCCTTATAG 12 7803 7821 6-7-6 696 599346 2569 2587 Exon 18 CAGCAGGAAACCCCTTATA 30 7804 7822 5-9-5 697 599288 2569 2587 Exon 18 CAGCAGGAAACCCCTTATA 28 7804 7822 6-7-6 697 599347 2570 2588 Exon 18 CCAGCAGGAAACCCCTTAT 46 7805 7823 5-9-5 698 599289 2570 2588 Exon 18 CCAGCAGGAAACCCCTTAT 32 7805 7823 6-7-6 698 599348 2571 2589 Exon 18 TCCAGCAGGAAACCCCTTA 44 7806 7824 5-9-5 699 599290 2571 2589 Exon 18 TCCAGCAGGAAACCCCTTA 24 7806 7824 6-7-6 699 599349 2572 2590 Exon 18 GTCCAGCAGGAAACCCCTT 60 7807 7825 5-9-5 700 599291 2572 2590 Exon 18 GTCCAGCAGGAAACCCCTT 38 7807 7825 6-7-6 700 599350 2573 2591 Exon 18 TGTCCAGCAGGAAACCCCT 49 7808 7826 5-9-5 701 599292 2573 2591 Exon 18 TGTCCAGCAGGAAACCCCT 35 7808 7826 6-7-6 701 599351 2575 2593 Exon 18 CCTGTCCAGCAGGAAACCC 46 7810 7828 5-9-5 702 599293 2575 2593 Exon 18 CCTGTCCAGCAGGAAACCC 12 7810 7828 6-7-6 702 599352 2576 2594 Exon 18 CCCTGTCCAGCAGGAAACC 49 7811 7829 5-9-5 703 599294 2576 2594 Exon 18 CCCTGTCCAGCAGGAAACC 38 7811 7829 6-7-6 703 599353 2577 2595 Exon 18 CCCCTGTCCAGCAGGAAAC 64 7812 7830 5-9-5 704 599295 2577 2595 Exon 18 CCCCTGTCCAGCAGGAAAC 33 7812 7830 6-7-6 704 599354 2578 2596 Exon 18 GCCCCTGTCCAGCAGGAAA 56 7813 7831 5-9-5 705 599296 2578 2596 Exon 18 GCCCCTGTCCAGCAGGAAA 13 7813 7831 6-7-6 705 599355 2580 2598 Exon 18 ACGCCCCTGTCCAGCAGGA 81 7815 7833 5-9-5 706 599297 2580 2598 Exon 18 ACGCCCCTGTCCAGCAGGA 57 7815 7833 6-7-6 706 599356 2581 2599 Exon 18 CACGCCCCTGTCCAGCAGG 64 7816 7834 5-9-5 707 599298 2581 2599 Exon 18 CACGCCCCTGTCCAGCAGG 39 7816 7834 6-7-6 707 599299 2582 2600 Exon 18 CCACGCCCCTGTCCAGCAG 55 7817 7835 6-7-6 708 599300 2583 2601 Exon 18 CCCACGCCCCTGTCCAGCA 45 7818 7836 6-7-6 709 599301 2584 2602 Exon 18 TCCCACGCCCCTGTCCAGC 39 7819 7837 6-7-6 710 599302 2585 2603 Exon 18 ATCCCACGCCCCTGTCCAG 27 7820 7838 6-7-6 711 599303 2586 2604 Exon 18 AATCCCACGCCCCTGTCCA 35 7821 7839 6-7-6 712 599304 2587 2605 Exon 18 CAATCCCACGCCCCTGTCC 16 7822 7840 6-7-6 713 599305 2588 2606 Exon 18 TCAATCCCACGCCCCTGTC 41 7823 7841 6-7-6 714 599306 2589 2607 Exon 18 TTCAATCCCACGCCCCTGT 70 7824 7842 6-7-6 715 599307 2590 2608 Exon 18 ATTCAATCCCACGCCCCTG 66 7825 7843 6-7-6 716 599308 2591 2609 Exon 18 AATTCAATCCCACGCCCCT 68 7826 7844 6-7-6 717 599309 2592 2610 Exon 18 TAATTCAATCCCACGCCCC 52 7827 7845 6-7-6 718 599310 2593 2611 Exon 18 TTAATTCAATCCCACGCCC 39 7828 7846 6-7-6 719 599311 2594 2612 Exon 18 TTTAATTCAATCCCACGCC 83 7829 7847 6-7-6 720 599312 2595 2613 Exon 18 TTTTAATTCAATCCCACGC 72 7830 7848 6-7-6 721 599313 2596 2614 Exon 18 GTTTTAATTCAATCCCACG 86 7831 7849 6-7-6 722 599314 2597 2615 Exon 18 TGTTTTAATTCAATCCCAC 91 7832 7850 6-7-6 723 599315 2598 2616 Exon 18 CTGTTTTAATTCAATCCCA 71 7833 7851 6-7-6 724 599316 2599 2617 Exon 18 GCTGTTTTAATTCAATCCC 89 7834 7852 6-7-6 725 599317 2600 2618 Exon 18 AGCTGTTTTAATTCAATCC 87 7835 7853 6-7-6 726 599318 2601 2619 Exon 18 CAGCTGTTTTAATTCAATC 81 7836 7854 6-7-6 727 599319 2602 2620 Exon 18 GCAGCTGTTTTAATTCAAT 75 7837 7855 6-7-6 728 599320 2603 2621 Exon 18 CGCAGCTGTTTTAATTCAA 84 7838 7856 6-7-6 729 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 92 7839 7858 5-10-5 317 599321 2604 2622 Exon 18 TCGCAGCTGTTTTAATTCA 90 7839 7857 6-7-6 730 599322 2605 2623 Exon 18 GTCGCAGCTGTTTTAATTC 89 7840 7858 6-7-6 731 599323 2606 2624 Exon 18 TGTCGCAGCTGTTTTAATT 81 7841 7859 6-7-6 732 599324 2607 2625 Exon 18 TTGTCGCAGCTGTTTTAAT 68 7842 7860 6-7-6 733 599325 2608 2626 Exon 18 GTTGTCGCAGCTGTTTTAA 71 7843 7861 6-7-6 734 599326 2609 2627 Exon 18 TGTTGTCGCAGCTGTTTTA 52 7844 7862 6-7-6 735 599327 2610 2628 Exon 18/ TTGTTGTCGCAGCTGTTTT 88 n/a n/a 6-7-6 736 Repeat 599328 2611 2629 Exon 18/ TTTGTTGTCGCAGCTGTTT 87 n/a n/a 6-7-6 737 Repeat 599329 2612 2630 Exon 18/ TTTTGTTGTCGCAGCTGTT 84 n/a n/a 6-7-6 738 Repeat 599330 2613 2631 Exon 18/ TTTTTGTTGTCGCAGCTGT 87 n/a n/a 6-7-6 739 Repeat

TABLE 137 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site Motif NO: 599512 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 74 7787 7806 3-10-7 410 599449 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 43 7788 7807 3-10-7 411 599450 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 51 7789 7808 3-10-7 412 599451 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 35 7790 7809 3-10-7 413 599452 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC 34 7791 7810 3-10-7 414 599453 2557 2576 Exon 18 CCCTTATAGAAAACCCAAAT 44 7792 7811 3-10-7 415 599454 2558 2577 Exon 18 CCCCTTATAGAAAACCCAAA 54 7793 7812 3-10-7 416 599455 2559 2578 Exon 18 ACCCCTTATAGAAAACCCAA 53 7794 7813 3-10-7 417 599456 2560 2579 Exon 18 AACCCCTTATAGAAAACCCA 69 7795 7814 3-10-7 418 599457 2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 46 7796 7815 3-10-7 419 599458 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 0 7797 7816 3-10-7 420 599459 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 12 7798 7817 3-10-7 421 599460 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 17 7799 7818 3-10-7 422 599461 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 24 7800 7819 3-10-7 423 599462 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA 33 7801 7820 3-10-7 424 599463 2567 2586 Exon 18 AGCAGGAAACCCCTTATAGA 38 7802 7821 3-10-7 425 599464 2568 2587 Exon 18 CAGCAGGAAACCCCTTATAG 33 7803 7822 3-10-7 426 599465 2569 2588 Exon 18 CCAGCAGGAAACCCCTTATA 49 7804 7823 3-10-7 427 599466 2570 2589 Exon 18 TCCAGCAGGAAACCCCTTAT 45 7805 7824 3-10-7 428 599467 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 60 7806 7825 3-10-7 237 599468 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 61 7807 7826 3-10-7 429 599469 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 52 7808 7827 3-10-7 430 599470 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 45 7809 7828 3-10-7 431 599471 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 67 7810 7829 3-10-7 432 599472 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC 79 7811 7830 3-10-7 433 599473 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 72 7812 7831 3-10-7 238 599474 2578 2597 Exon 18 CGCCCCTGTCCAGCAGGAAA 87 7813 7832 3-10-7 434 599475 2579 2598 Exon 18 ACGCCCCTGTCCAGCAGGAA 76 7814 7833 3-10-7 435 599476 2580 2599 Exon 18 CACGCCCCTGTCCAGCAGGA 81 7815 7834 3-10-7 436 599477 2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 83 7816 7835 3-10-7 437 599478 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 72 7817 7836 3-10-7 438 599479 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 81 7818 7837 3-10-7 439 599480 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 77 7819 7838 3-10-7 440 599481 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 83 7820 7839 3-10-7 441 599482 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA 87 7821 7840 3-10-7 442 599483 2587 2606 Exon 18 TCAATCCCACGCCCCTGTCC 90 7822 7841 3-10-7 443 599484 2588 2607 Exon 18 TTCAATCCCACGCCCCTGTC 72 7823 7842 3-10-7 444 599485 2589 2608 Exon 18 ATTCAATCCCACGCCCCTGT 82 7824 7843 3-10-7 445 599486 2590 2609 Exon 18 AATTCAATCCCACGCCCCTG 84 7825 7844 3-10-7 446 599487 2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 84 7826 7845 3-10-7 447 599488 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 87 7827 7846 3-10-7 448 599489 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 87 7828 7847 3-10-7 449 599490 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 86 7829 7848 3-10-7 450 599491 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 87 7830 7849 3-10-7 451 599492 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG 88 7831 7850 3-10-7 452 599493 2597 2616 Exon 18 CTGTTTTAATTCAATCCCAC 75 7832 7851 3-10-7 453 599433 2597 2616 Exon 18 CTGTTTTAATTCAATCCCAC 89 7832 7851 6-8-6 453 599494 2598 2617 Exon 18 GCTGTTTTAATTCAATCCCA 90 7833 7852 3-10-7 454 599434 2598 2617 Exon 18 GCTGTTTTAATTCAATCCCA 89 7833 7852 6-8-6 454 599495 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 88 7834 7853 3-10-7 239 599435 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 91 7834 7853 6-8-6 239 599496 2600 2619 Exon 18 CAGCTGTTTTAATTCAATCC 89 7835 7854 3-10-7 455 599436 2600 2619 Exon 18 CAGCTGTTTTAATTCAATCC 89 7835 7854 6-8-6 455 599497 2601 2620 Exon 18 GCAGCTGTTTTAATTCAATC 89 7836 7855 3-10-7 456 599437 2601 2620 Exon 18 GCAGCTGTTTTAATTCAATC 91 7836 7855 6-8-6 456 599498 2602 2621 Exon 18 CGCAGCTGTTTTAATTCAAT 88 7837 7856 3-10-7 457 599438 2602 2621 Exon 18 CGCAGCTGTTTTAATTCAAT 90 7837 7856 6-8-6 457 599499 2603 2622 Exon 18 TCGCAGCTGTTTTAATTCAA 81 7838 7857 3-10-7 458 599439 2603 2622 Exon 18 TCGCAGCTGTTTTAATTCAA 88 7838 7857 6-8-6 458 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 90 7839 7858 5-10-5 317 599500 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 88 7839 7858 3-10-7 317 599440 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 88 7839 7858 6-8-6 317 599501 2605 2624 Exon 18 TGTCGCAGCTGTTTTAATTC 78 7840 7859 3-10-7 459 599441 2605 2624 Exon 18 TGTCGCAGCTGTTTTAATTC 90 7840 7859 6-8-6 459 599502 2606 2625 Exon 18 TTGTCGCAGCTGTTTTAATT 87 7841 7860 3-10-7 460 599442 2606 2625 Exon 18 TTGTCGCAGCTGTTTTAATT 76 7841 7860 6-8-6 460 599503 2607 2626 Exon 18 GTTGTCGCAGCTGTTTTAAT 83 7842 7861 3-10-7 461 599443 2607 2626 Exon 18 GTTGTCGCAGCTGTTTTAAT 77 7842 7861 6-8-6 461 599504 2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 89 7843 7862 3-10-7 395 599444 2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 69 7843 7862 6-8-6 395 599505 2609 2628 Exon 19/ TTGTTGTCGCAGCTGTTTTA 83 n/a n/a 3-10-7 462 Repeat 599445 2609 2628 Exon 19/ TTGTTGTCGCAGCTGTTTTA 85 n/a n/a 6-8-6 462 Repeat 599506 2610 2629 Exon 19/ TTTGTTGTCGCAGCTGTTTT 89 n/a n/a 3-10-7 463 Repeat 599446 2610 2629 Exon 19/ TTTGTTGTCGCAGCTGTTTT 85 n/a n/a 6-8-6 463 Repeat 599507 2611 2630 Exon 19/ TTTTGTTGTCGCAGCTGTTT 82 n/a n/a 3-10-7 464 Repeat 599447 2611 2630 Exon 19/ TTTTGTTGTCGCAGCTGTTT 83 n/a n/a 6-8-6 464 Repeat 599508 2612 2631 Exon 19/ TTTTTGTTGTCGCAGCTGTT 90 n/a n/a 3-10-7 465 Repeat 599448 2612 2631 Exon 19/ TTTTTGTTGTCGCAGCTGTT 87 n/a n/a 6-8-6 465 Repeat

Example 119: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 4-8-5 MOE, 5-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE, 6-7-6-MOE, 3-10-5 MOE, or 6-8-6 MOE gapmers.

The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four and five nucleosides respectively. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and five nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 138 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site Motif NO: 599160 2560 2577 Exon 18 CCCCTTATAGAAAACCCA 26 7795 7812 5-8-5 740 599161 2561 2578 Exon 18 ACCCCTTATAGAAAACCC 20 7796 7813 5-8-5 741 599162 2562 2579 Exon 18 AACCCCTTATAGAAAACC 12 7797 7814 5-8-5 742 599163 2563 2580 Exon 18 AAACCCCTTATAGAAAAC 11 7798 7815 5-8-5 743 599164 2564 2581 Exon 18 GAAACCCCTTATAGAAAA 11 7799 7816 5-8-5 744 599165 2566 2583 Exon 18 AGGAAACCCCTTATAGAA 0 7801 7818 5-8-5 745 599166 2567 2584 Exon 18 CAGGAAACCCCTTATAGA 12 7802 7819 5-8-5 746 599167 2568 2585 Exon 18 GCAGGAAACCCCTTATAG 14 7803 7820 5-8-5 747 599168 2569 2586 Exon 18 AGCAGGAAACCCCTTATA 16 7804 7821 5-8-5 748 599169 2570 2587 Exon 18 CAGCAGGAAACCCCTTAT 24 7805 7822 5-8-5 749 599170 2571 2588 Exon 18 CCAGCAGGAAACCCCTTA 37 7806 7823 5-8-5 750 599171 2572 2589 Exon 18 TCCAGCAGGAAACCCCTT 30 7807 7824 5-8-5 751 599172 2573 2590 Exon 18 GTCCAGCAGGAAACCCCT 43 7808 7825 5-8-5 752 599173 2574 2591 Exon 18 TGTCCAGCAGGAAACCCC 47 7809 7826 5-8-5 753 599174 2575 2592 Exon 18 CTGTCCAGCAGGAAACCC 27 7810 7827 5-8-5 754 599175 2576 2593 Exon 18 CCTGTCCAGCAGGAAACC 30 7811 7828 5-8-5 755 599176 2577 2594 Exon 18 CCCTGTCCAGCAGGAAAC 34 7812 7829 5-8-5 756 599177 2578 2595 Exon 18 CCCCTGTCCAGCAGGAAA 41 7813 7830 5-8-5 757 599178 2580 2597 Exon 18 CGCCCCTGTCCAGCAGGA 67 7815 7832 5-8-5 758 599179 2581 2598 Exon 18 ACGCCCCTGTCCAGCAGG 61 7816 7833 5-8-5 759 599180 2582 2599 Exon 18 CACGCCCCTGTCCAGCAG 62 7817 7834 5-8-5 760 599181 2583 2600 Exon 18 CCACGCCCCTGTCCAGCA 63 7818 7835 5-8-5 761 599128 2584 2600 Exon 18 CCACGCCCCTGTCCAGC 55 7819 7835 4-8-5 649 599182 2584 2601 Exon 18 CCCACGCCCCTGTCCAGC 58 7819 7836 5-8-5 762 599129 2585 2601 Exon 18 CCCACGCCCCTGTCCAG 41 7820 7836 4-8-5 650 599183 2585 2602 Exon 18 TCCCACGCCCCTGTCCAG 43 7820 7837 5-8-5 763 599130 2586 2602 Exon 18 TCCCACGCCCCTGTCCA 46 7821 7837 4-8-5 651 599184 2586 2603 Exon 18 ATCCCACGCCCCTGTCCA 32 7821 7838 5-8-5 764 599131 2587 2603 Exon 18 ATCCCACGCCCCTGTCC 30 7822 7838 4-8-5 652 599185 2587 2604 Exon 18 AATCCCACGCCCCTGTCC 35 7822 7839 5-8-5 765 599132 2588 2604 Exon 18 AATCCCACGCCCCTGTC 52 7823 7839 4-8-5 653 599186 2588 2605 Exon 18 CAATCCCACGCCCCTGTC 55 7823 7840 5-8-5 766 599133 2589 2605 Exon 18 CAATCCCACGCCCCTGT 66 7824 7840 4-8-5 654 599187 2589 2606 Exon 18 TCAATCCCACGCCCCTGT 72 7824 7841 5-8-5 767 599134 2590 2606 Exon 18 TCAATCCCACGCCCCTG 80 7825 7841 4-8-5 655 599188 2590 2607 Exon 18 TTCAATCCCACGCCCCTG 92 7825 7842 5-8-5 768 599135 2591 2607 Exon 18 TTCAATCCCACGCCCCT 61 7826 7842 4-8-5 656 599189 2591 2608 Exon 18 ATTCAATCCCACGCCCCT 52 7826 7843 5-8-5 769 599136 2592 2608 Exon 18 ATTCAATCCCACGCCCC 68 7827 7843 4-8-5 657 599190 2592 2609 Exon 18 AATTCAATCCCACGCCCC 62 7827 7844 5-8-5 770 599137 2593 2609 Exon 18 AATTCAATCCCACGCCC 51 7828 7844 4-8-5 658 599191 2593 2610 Exon 18 TAATTCAATCCCACGCCC 54 7828 7845 5-8-5 771 599138 2594 2610 Exon 18 TAATTCAATCCCACGCC 71 7829 7845 4-8-5 659 599192 2594 2611 Exon 18 TTAATTCAATCCCACGCC 66 7829 7846 5-8-5 772 599139 2595 2611 Exon 18 TTAATTCAATCCCACGC 80 7830 7846 4-8-5 660 599193 2595 2612 Exon 18 TTTAATTCAATCCCACGC 74 7830 7847 5-8-5 773 599140 2596 2612 Exon 18 TTTAATTCAATCCCACG 66 7831 7847 4-8-5 786 599194 2596 2613 Exon 18 TTTTAATTCAATCCCACG 66 7831 7848 5-8-5 774 599141 2597 2613 Exon 18 TTTTAATTCAATCCCAC 63 7832 7848 4-8-5 662 599195 2597 2614 Exon 18 GTTTTAATTCAATCCCAC 86 7832 7849 5-8-5 775 599142 2598 2614 Exon 18 GTTTTAATTCAATCCCA 69 7833 7849 4-8-5 663 599196 2598 2615 Exon 18 TGTTTTAATTCAATCCCA 82 7833 7850 5-8-5 776 599143 2599 2615 Exon 18 TGTTTTAATTCAATCCC 59 7834 7850 4-8-5 664 599197 2599 2616 Exon 18 CTGTTTTAATTCAATCCC 79 7834 7851 5-8-5 777 599144 2600 2616 Exon 18 CTGTTTTAATTCAATCC 52 7835 7851 4-8-5 665 599198 2600 2617 Exon 18 GCTGTTTTAATTCAATCC 86 7835 7852 5-8-5 778 599145 2601 2617 Exon 18 GCTGTTTTAATTCAATC 53 7836 7852 4-8-5 666 599199 2601 2618 Exon 18 AGCTGTTTTAATTCAATC 72 7836 7853 5-8-5 779 599146 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 42 7837 7853 4-8-5 667 599200 2602 2619 Exon 18 CAGCTGTTTTAATTCAAT 76 7837 7854 5-8-5 780 599147 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 55 7838 7854 4-8-5 668 599201 2603 2620 Exon 18 GCAGCTGTTTTAATTCAA 87 7838 7855 5-8-5 781 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 93 7839 7858 5-10-5 317 599148 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 84 7839 7855 4-8-5 669 599202 2604 2621 Exon 18 CGCAGCTGTTTTAATTCA 89 7839 7856 5-8-5 782 599149 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 92 7840 7856 4-8-5 670 599203 2605 2622 Exon 18 TCGCAGCTGTTTTAATTC 90 7840 7857 5-8-5 783 599150 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 75 7841 7857 4-8-5 671 599151 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 80 7842 7858 4-8-5 672 599152 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 76 7843 7859 4-8-5 673 599153 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 56 7844 7860 4-8-5 674 599154 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 85 7845 7861 4-8-5 675 599155 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 89 7846 7862 4-8-5 676 599156 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 83 n/a n/a 4-8-5 813 Repeat 599157 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 78 n/a n/a 4-8-5 678 Repeat 599158 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 83 n/a n/a 4-8-5 679 Repeat 599159 2615 2631 Exon 18/ TTTTTGTTGTCGCAGCT 65 n/a n/a 4-8-5 680 Repeat 599204 2606 2623 Exon 18 GTCGCAGCTGTTTTAATT 83 7841 7858 5-8-5 784

TABLE 139 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ start stop Target % start stop ID ISIS NO site site region Sequence inhibition site site Motif NO: 599509 2552 2570 Exon 18 TAGAAAACCCAAATCCTCA 45 7787 7805 6-7-6 681 599213 2553 2570 Exon 18 TAGAAAACCCAAATCCTC 89 7788 7805 3-10-5 785 599273 2553 2571 Exon 18 ATAGAAAACCCAAATCCTC 85 7788 7806 6-7-6 682 599214 2554 2571 Exon 18 ATAGAAAACCCAAATCCT 79 7789 7806 3-10-5 786 599274 2554 2572 Exon 18 TATAGAAAACCCAAATCCT 75 7789 7807 6-7-6 683 599215 2555 2572 Exon 18 TATAGAAAACCCAAATCC 81 7790 7807 3-10-5 787 599216 2556 2573 Exon 18 TTATAGAAAACCCAAATC 87 7791 7808 3-10-5 788 599275 2556 2574 Exon 18 CTTATAGAAAACCCAAATC 84 7791 7809 6-7-6 684 599217 2557 2574 Exon 18 CTTATAGAAAACCCAAAT 84 7792 7809 3-10-5 789 599276 2557 2575 Exon 18 CCTTATAGAAAACCCAAAT 68 7792 7810 6-7-6 685 599218 2558 2575 Exon 18 CCTTATAGAAAACCCAAA 82 7793 7810 3-10-5 790 599277 2558 2576 Exon 18 CCCTTATAGAAAACCCAAA 82 7793 7811 6-7-6 686 599219 2559 2576 Exon 18 CCCTTATAGAAAACCCAA 81 7794 7811 3-10-5 791 599278 2559 2577 Exon 18 CCCCTTATAGAAAACCCAA 84 7794 7812 6-7-6 687 599220 2560 2577 Exon 18 CCCCTTATAGAAAACCCA 92 7795 7812 3-10-5 740 599279 2560 2578 Exon 18 ACCCCTTATAGAAAACCCA 92 7795 7813 6-7-6 688 599221 2561 2578 Exon 18 ACCCCTTATAGAAAACCC 93 7796 7813 3-10-5 741 599280 2561 2579 Exon 18 AACCCCTTATAGAAAACCC 90 7796 7814 6-7-6 689 599222 2562 2579 Exon 18 AACCCCTTATAGAAAACC 95 7797 7814 3-10-5 742 599223 2563 2580 Exon 18 AAACCCCTTATAGAAAAC 93 7798 7815 3-10-5 743 599224 2564 2581 Exon 18 GAAACCCCTTATAGAAAA 90 7799 7816 3-10-5 744 599225 2566 2583 Exon 18 AGGAAACCCCTTATAGAA 93 7801 7818 3-10-5 745 599226 2567 2584 Exon 18 CAGGAAACCCCTTATAGA 95 7802 7819 3-10-5 746 599227 2568 2585 Exon 18 GCAGGAAACCCCTTATAG 94 7803 7820 3-10-5 747 599228 2569 2586 Exon 18 AGCAGGAAACCCCTTATA 96 7804 7821 3-10-5 748 599229 2570 2587 Exon 18 CAGCAGGAAACCCCTTAT 92 7805 7822 3-10-5 749 599230 2571 2588 Exon 18 CCAGCAGGAAACCCCTTA 88 7806 7823 3-10-5 750 599231 2572 2589 Exon 18 TCCAGCAGGAAACCCCTT 83 7807 7824 3-10-5 751 599232 2573 2590 Exon 18 GTCCAGCAGGAAACCCCT 89 7808 7825 3-10-5 752 599233 2574 2591 Exon 18 TGTCCAGCAGGAAACCCC 83 7809 7826 3-10-5 753 599234 2575 2592 Exon 18 CTGTCCAGCAGGAAACCC 88 7810 7827 3-10-5 754 599235 2576 2593 Exon 18 CCTGTCCAGCAGGAAACC 91 7811 7828 3-10-5 755 599236 2577 2594 Exon 18 CCCTGTCCAGCAGGAAAC 90 7812 7829 3-10-5 756 599237 2578 2595 Exon 18 CCCCTGTCCAGCAGGAAA 34 7813 7830 3-10-5 757 599238 2580 2597 Exon 18 CGCCCCTGTCCAGCAGGA 14 7815 7832 3-10-5 758 599239 2581 2598 Exon 18 ACGCCCCTGTCCAGCAGG 10 7816 7833 3-10-5 759 599240 2582 2599 Exon 18 CACGCCCCTGTCCAGCAG 26 7817 7834 3-10-5 760 599241 2583 2600 Exon 18 CCACGCCCCTGTCCAGCA 11 7818 7835 3-10-5 761 599242 2584 2601 Exon 18 CCCACGCCCCTGTCCAGC 24 7819 7836 3-10-5 762 599243 2585 2602 Exon 18 TCCCACGCCCCTGTCCAG 23 7820 7837 3-10-5 763 599244 2586 2603 Exon 18 ATCCCACGCCCCTGTCCA 29 7821 7838 3-10-5 764 599245 2587 2604 Exon 18 AATCCCACGCCCCTGTCC 11 7822 7839 3-10-5 765 599246 2588 2605 Exon 18 CAATCCCACGCCCCTGTC 0 7823 7840 3-10-5 766 599247 2589 2606 Exon 18 TCAATCCCACGCCCCTGT 21 7824 7841 3-10-5 767 599248 2590 2607 Exon 18 TTCAATCCCACGCCCCTG 0 7825 7842 3-10-5 768 599249 2591 2608 Exon 18 ATTCAATCCCACGCCCCT 9 7826 7843 3-10-5 769 599250 2592 2609 Exon 18 AATTCAATCCCACGCCCC 4 7827 7844 3-10-5 770 599251 2593 2610 Exon 18 TAATTCAATCCCACGCCC 12 7828 7845 3-10-5 771 599252 2594 2611 Exon 18 TTAATTCAATCCCACGCC 2 7829 7846 3-10-5 772 599253 2595 2612 Exon 18 TTTAATTCAATCCCACGC 28 7830 7847 3-10-5 773 599254 2596 2613 Exon 18 TTTTAATTCAATCCCACG 27 7831 7848 3-10-5 774 599255 2597 2614 Exon 18 GTTTTAATTCAATCCCAC 38 7832 7849 3-10-5 775 599256 2598 2615 Exon 18 TGTTTTAATTCAATCCCA 36 7833 7850 3-10-5 776 599257 2599 2616 Exon 18 CTGTTTTAATTCAATCCC 48 7834 7851 3-10-5 777 599258 2600 2617 Exon 18 GCTGTTTTAATTCAATCC 19 7835 7852 3-10-5 778 599259 2601 2618 Exon 18 AGCTGTTTTAATTCAATC 36 7836 7853 3-10-5 779 599260 2602 2619 Exon 18 CAGCTGTTTTAATTCAAT 58 7837 7854 3-10-5 780 599261 2603 2620 Exon 18 GCAGCTGTTTTAATTCAA 35 7838 7855 3-10-5 781 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 96 7839 7858 5-10-5 317 599262 2604 2621 Exon 18 CGCAGCTGTTTTAATTCA 52 7839 7856 3-10-5 782 599263 2605 2622 Exon 18 TCGCAGCTGTTTTAATTC 66 7840 7857 3-10-5 783 599264 2606 2623 Exon 18 GTCGCAGCTGTTTTAATT 48 7841 7858 3-10-5 784 599265 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 46 7842 7859 3-10-5 792 599205 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 83 7842 7859 5-8-5 792 599266 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 76 7843 7860 3-10-5 793 599206 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 90 7843 7860 5-8-5 793 599267 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 53 7844 7861 3-10-5 794 599207 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 82 7844 7861 5-8-5 794 599268 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 58 7845 7862 3-10-5 795 599208 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 70 7845 7862 5-8-5 795 599269 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 38 n/a n/a 3-10-5 796 Repeat 599209 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 50 n/a n/a 5-8-5 796 Repeat 599270 2612 2629 Exon 18/ TTTGTTGTCGCAGCTGTT 46 n/a n/a 3-10-5 797 Repeat 599210 2612 2629 Exon 18/ TTTGTTGTCGCAGCTGTT 76 n/a n/a 5-8-5 797 Repeat 599271 2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 64 n/a n/a 3-10-5 798 Repeat 599211 2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 78 n/a n/a 5-8-5 798 Repeat 599272 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 89 n/a n/a 3-10-5 799 Repeat 599212 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 84 n/a n/a 5-8-5 799 Repeat

TABLE 140 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site Motif NO: 599511 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 38 7787 7806 6-8-6 410 599389 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 80 7788 7807 6-8-6 411 599390 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 92 7789 7808 6-8-6 412 599391 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 90 7790 7809 6-8-6 413 599392 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC 87 7791 7810 6-8-6 414 599393 2557 2576 Exon 18 CCCTTATAGAAAACCCAAAT 87 7792 7811 6-8-6 415 599394 2558 2577 Exon 18 CCCCTTATAGAAAACCCAAA 74 7793 7812 6-8-6 416 599395 2559 2578 Exon 18 ACCCCTTATAGAAAACCCAA 78 7794 7813 6-8-6 417 599396 2560 2579 Exon 18 AACCCCTTATAGAAAACCCA 77 7795 7814 6-8-6 418 599397 2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 89 7796 7815 6-8-6 419 599398 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 90 7797 7816 6-8-6 420 599399 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 91 7798 7817 6-8-6 421 599400 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 88 7799 7818 6-8-6 422 599401 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 85 7800 7819 6-8-6 423 599402 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA 77 7801 7820 6-8-6 424 599403 2567 2586 Exon 18 AGCAGGAAACCCCTTATAGA 85 7802 7821 6-8-6 425 599404 2568 2587 Exon 18 CAGCAGGAAACCCCTTATAG 90 7803 7822 6-8-6 426 599405 2569 2588 Exon 18 CCAGCAGGAAACCCCTTATA 89 7804 7823 6-8-6 427 599406 2570 2589 Exon 18 TCCAGCAGGAAACCCCTTAT 72 7805 7824 6-8-6 428 599407 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 87 7806 7825 6-8-6 237 599408 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 87 7807 7826 6-8-6 429 599409 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 83 7808 7827 6-8-6 430 599410 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 88 7809 7828 6-8-6 431 599411 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 45 7810 7829 6-8-6 432 599412 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC 66 7811 7830 6-8-6 433 599413 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 92 7812 7831 6-8-6 238 599414 2578 2597 Exon 18 CGCCCCTGTCCAGCAGGAAA 92 7813 7832 6-8-6 434 599415 2579 2598 Exon 18 ACGCCCCTGTCCAGCAGGAA 87 7814 7833 6-8-6 435 599416 2580 2599 Exon 18 CACGCCCCTGTCCAGCAGGA 91 7815 7834 6-8-6 436 599417 2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 84 7816 7835 6-8-6 437 599357 2582 2600 Exon 18 CCACGCCCCTGTCCAGCAG 88 7817 7835 5-9-5 708 599418 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 85 7817 7836 6-8-6 438 599358 2583 2601 Exon 18 CCCACGCCCCTGTCCAGCA 86 7818 7836 5-9-5 709 599419 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 91 7818 7837 6-8-6 833 599359 2584 2602 Exon 18 TCCCACGCCCCTGTCCAGC 85 7819 7837 5-9-5 834 599420 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 91 7819 7838 6-8-6 440 599360 2585 2603 Exon 18 ATCCCACGCCCCTGTCCAG 89 7820 7838 5-9-5 711 599421 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 87 7820 7839 6-8-6 441 599361 2586 2604 Exon 18 AATCCCACGCCCCTGTCCA 89 7821 7839 5-9-5 712 599422 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA 90 7821 7840 6-8-6 442 599362 2587 2605 Exon 18 CAATCCCACGCCCCTGTCC 94 7822 7840 5-9-5 713 599423 2587 2606 Exon 18 TCAATCCCACGCCCCTGTCC 85 7822 7841 6-8-6 841 599363 2588 2606 Exon 18 TCAATCCCACGCCCCTGTC 88 7823 7841 5-9-5 714 599424 2588 2607 Exon 18 TTCAATCCCACGCCCCTGTC 88 7823 7842 6-8-6 444 599364 2589 2607 Exon 18 TTCAATCCCACGCCCCTGT 88 7824 7842 5-9-5 715 599425 2589 2608 Exon 18 ATTCAATCCCACGCCCCTGT 68 7824 7843 6-8-6 445 599365 2590 2608 Exon 18 ATTCAATCCCACGCCCCTG 48 7825 7843 5-9-5 716 599426 2590 2609 Exon 18 AATTCAATCCCACGCCCCTG 55 7825 7844 6-8-6 446 599366 2591 2609 Exon 18 AATTCAATCCCACGCCCCT 28 7826 7844 5-9-5 717 599427 2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 13 7826 7845 6-8-6 849 599367 2592 2610 Exon 18 TAATTCAATCCCACGCCCC 21 7827 7845 5-9-5 718 599428 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 39 7827 7846 6-8-6 448 599368 2593 2611 Exon 18 TTAATTCAATCCCACGCCC 20 7828 7846 5-9-5 719 599429 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 18 7828 7847 6-8-6 449 599369 2594 2612 Exon 18 TTTAATTCAATCCCACGCC 78 7829 7847 5-9-5 720 599430 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 24 7829 7848 6-8-6 450 599370 2595 2613 Exon 18 TTTTAATTCAATCCCACGC 25 7830 7848 5-9-5 721 599431 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 30 7830 7849 6-8-6 451 599371 2596 2614 Exon 18 GTTTTAATTCAATCCCACG 84 7831 7849 5-9-5 722 599432 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG 29 7831 7850 6-8-6 452 599372 2597 2615 Exon 18 TGTTTTAATTCAATCCCAC 83 7832 7850 5-9-5 723 599373 2598 2616 Exon 18 CTGTTTTAATTCAATCCCA 81 7833 7851 5-9-5 724 599374 2599 2617 Exon 18 GCTGTTTTAATTCAATCCC 26 7834 7852 5-9-5 725 599375 2600 2618 Exon 18 AGCTGTTTTAATTCAATCC 26 7835 7853 5-9-5 726 599376 2601 2619 Exon 18 CAGCTGTTTTAATTCAATC 62 7836 7854 5-9-5 727 599377 2602 2620 Exon 18 GCAGCTGTTTTAATTCAAT 21 7837 7855 5-9-5 728 599378 2603 2621 Exon 18 CGCAGCTGTTTTAATTCAA 90 7838 7856 5-9-5 729 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 95 7839 7858 5-10-5 867 599379 2604 2622 Exon 18 TCGCAGCTGTTTTAATTCA 88 7839 7857 5-9-5 730 599380 2605 2623 Exon 18 GTCGCAGCTGTTTTAATTC 37 7840 7858 5-9-5 869 599381 2606 2624 Exon 18 TGTCGCAGCTGTTTTAATT 33 7841 7859 5-9-5 732 599382 2607 2625 Exon 18 TTGTCGCAGCTGTTTTAAT 81 7842 7860 5-9-5 733 599383 2608 2626 Exon 18 GTTGTCGCAGCTGTTTTAA 54 7843 7861 5-9-5 734 599384 2609 2627 Exon 18 TGTTGTCGCAGCTGTTTTA 85 7844 7862 5-9-5 873 599385 2610 2628 Exon 18/ TTGTTGTCGCAGCTGTTTT 59 n/a n/a 5-9-5 736 Repeat 599386 2611 2629 Exon 18/ TTTGTTGTCGCAGCTGTTT 81 n/a n/a 5-9-5 737 Repeat 599387 2612 2630 Exon 18/ TTTTGTTGTCGCAGCTGTT 80 n/a n/a 5-9-5 738 Repeat 599388 2613 2631 Exon 18/ TTTTTGTTGTCGCAGCTGT 84 n/a n/a 5-9-5 739 Repeat

Example 120: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed deoxy, MOE and (S)-cEt oligonucleotides. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 141 Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ start stop Target inhi- start stop ID ISIS NO site site region Sequence bition site site Motif NO: 599513 2551 2566 Exon 18 AAACCCAAATCCTCAT 11 7786 7801 ekkeekkdddddddkk 557 599514 2553 2568 Exon 18 GAAAACCCAAATCCTC 13 7788 7803 ekkeekkdddddddkk 801 599515 2555 2570 Exon 18 TAGAAAACCCAAATCC 54 7790 7805 ekkeekkdddddddkk 559 599516 2559 2574 Exon 18 CTTATAGAAAACCCAA 16 7794 7809 ekkeekkdddddddkk 561 599517 2560 2575 Exon 18 CCTTATAGAAAACCCA 29 7795 7810 ekkeekkdddddddkk 562 599518 2561 2576 Exon 18 CCCTTATAGAAAACCC 55 7796 7811 ekkeekkdddddddkk 563 599519 2562 2577 Exon 18 CCCCTTATAGAAAACC 31 7797 7812 ekkeekkdddddddkk 564 599520 2563 2578 Exon 18 ACCCCTTATAGAAAAC 14 7798 7813 ekkeekkdddddddkk 565 599521 2564 2579 Exon 18 AACCCCTTATAGAAAA 9 7799 7814 ekkeekkdddddddkk 566 599522 2565 2580 Exon 18 AAACCCCTTATAGAAA 8 7800 7815 ekkeekkdddddddkk 567 599523 2566 2581 Exon 18 GAAACCCCTTATAGAA 6 7801 7816 ekkeekkdddddddkk 568 599524 2567 2582 Exon 18 GGAAACCCCTTATAGA 14 7802 7817 ekkeekkdddddddkk 569 599525 2568 2583 Exon 18 AGGAAACCCCTTATAG 6 7803 7818 ekkeekkdddddddkk 570 599526 2569 2584 Exon 18 CAGGAAACCCCTTATA 16 7804 7819 ekkeekkdddddddkk 571 599527 2570 2585 Exon 18 GCAGGAAACCCCTTAT 0 7805 7820 ekkeekkdddddddkk 572 599528 2571 2586 Exon 18 AGCAGGAAACCCCTTA 6 7806 7821 ekkeekkdddddddkk 573 599529 2572 2587 Exon 18 CAGCAGGAAACCCCTT 6 7807 7822 ekkeekkdddddddkk 574 599530 2574 2589 Exon 18 TCCAGCAGGAAACCCC 29 7809 7824 ekkeekkdddddddkk 576 599531 2575 2590 Exon 18 GTCCAGCAGGAAACCC 64 7810 7825 ekkeekkdddddddkk 577 599532 2576 2591 Exon 18 TGTCCAGCAGGAAACC 43 7811 7826 ekkeekkdddddddkk 578 599533 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 25 7812 7827 ekkeekkdddddddkk 820 599534 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 12 7813 7828 ekkeekkdddddddkk 580 599535 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 16 7815 7830 ekkeekkdddddddkk 582 599536 2582 2597 Exon 18 CGCCCCTGTCCAGCAG 27 7817 7832 ekkeekkdddddddkk 584 599537 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 35 7818 7833 ekkeekkdddddddkk 585 599538 2584 2599 Exon 18 CACGCCCCTGTCCAGC 26 7819 7834 ekkeekkdddddddkk 586 599539 2585 2600 Exon 18 CCACGCCCCTGTCCAG 33 7820 7835 ekkeekkdddddddkk 587 599540 2586 2601 Exon 18 CCCACGCCCCTGTCCA 27 7821 7836 ekkeekkdddddddkk 588 599541 2587 2602 Exon 18 TCCCACGCCCCTGTCC 52 7822 7837 ekkeekkdddddddkk 589 599542 2588 2603 Exon 18 ATCCCACGCCCCTGTC 16 7823 7838 ekkeekkdddddddkk 590 599543 2589 2604 Exon 18 AATCCCACGCCCCTGT 19 7824 7839 ekkeekkdddddddkk 591 599544 2590 2605 Exon 18 CAATCCCACGCCCCTG 33 7825 7840 ekkeekkdddddddkk 831 599545 2591 2606 Exon 18 TCAATCCCACGCCCCT 24 7826 7841 ekkeekkdddddddkk 593 599546 2592 2607 Exon 18 TTCAATCCCACGCCCC 54 7827 7842 ekkeekkdddddddkk 594 599547 2593 2608 Exon 18 ATTCAATCCCACGCCC 87 7828 7843 ekkeekkdddddddkk 595 599548 2594 2609 Exon 18 AATTCAATCCCACGCC 79 7829 7844 ekkeekkdddddddkk 596 599549 2595 2610 Exon 18 TAATTCAATCCCACGC 62 7830 7845 ekkeekkdddddddkk 597 599550 2596 2611 Exon 18 TTAATTCAATCCCACG 52 7831 7846 ekkeekkdddddddkk 598 599551 2597 2612 Exon 18 TTTAATTCAATCCCAC 27 7832 7847 ekkeekkdddddddkk 599 599577 2597 2613 Exon 18 TTTTAATTCAATCCCAC 90 7832 7848 eeekkdddddddkkeee 662 599552 2598 2613 Exon 18 TTTTAATTCAATCCCA 92 7833 7848 ekkeekkdddddddkk 600 599578 2598 2614 Exon 18 GTTTTAATTCAATCCCA 88 7833 7849 eeekkdddddddkkeee 663 599553 2599 2614 Exon 18 GTTTTAATTCAATCCC 91 7834 7849 ekkeekkdddddddkk 601 599579 2599 2615 Exon 18 TGTTTTAATTCAATCCC 79 7834 7850 eeekkdddddddkkeee 664 599554 2600 2615 Exon 18 TGTTTTAATTCAATCC 90 7835 7850 ekkeekkdddddddkk 602 599580 2600 2616 Exon 18 CTGTTTTAATTCAATCC 79 7835 7851 eeekkdddddddkkeee 665 599555 2601 2616 Exon 18 CTGTTTTAATTCAATC 79 7836 7851 ekkeekkdddddddkk 846 599581 2601 2617 Exon 18 GCTGTTTTAATTCAATC 90 7836 7852 eeekkdddddddkkeee 666 599556 2602 2617 Exon 18 GCTGTTTTAATTCAAT 47 7837 7852 ekkeekkdddddddkk 604 599582 2602 2618 Exon 18 AGCTGTTTTAATTCAAT 89 7837 7853 eeekkdddddddkkeee 849 599557 2603 2618 Exon 18 AGCTGTTTTAATTCAA 67 7838 7853 ekkeekkdddddddkk 850 599583 2603 2619 Exon 18 CAGCTGTTTTAATTCAA 49 7838 7854 eeekkdddddddkkeee 668 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 78 7839 7858 eeeeeddddddddddeee 317 ee 599558 2604 2619 Exon 18 CAGCTGTTTTAATTCA 80 7839 7854 ekkeekkdddddddkk 606 599584 2604 2620 Exon 18 GCAGCTGTTTTAATTCA 66 7839 7855 eeekkdddddddkkeee 669 599559 2605 2620 Exon 18 GCAGCTGTTTTAATTC 38 7840 7855 ekkeekkdddddddkk 607 599585 2605 2621 Exon 18 CGCAGCTGTTTTAATTC 80 7840 7856 eeekkdddddddkkeee 670 599560 2606 2621 Exon 18 CGCAGCTGTTTTAATT 16 7841 7856 ekkeekkdddddddkk 608 599586 2606 2622 Exon 18 TCGCAGCTGTTTTAATT 78 7841 7857 eeekkdddddddkkeee 671 599561 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 58 7842 7857 ekkeekkdddddddkk 609 599587 2607 2623 Exon 18 GTCGCAGCTGTTTTAAT 81 7842 7858 eeekkdddddddkkeee 672 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 92 7843 7858 eekddddddddddkke 610 599562 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 78 7843 7858 ekkeekkdddddddkk 610 599588 2608 2624 Exon 18 TGTCGCAGCTGTTTTAA 81 7843 7859 eeekkdddddddkkeee 673 599563 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 86 7844 7859 ekkeekkdddddddkk 611 599589 2609 2625 Exon 18 TTGTCGCAGCTGTTTTA 75 7844 7860 eeekkdddddddkkeee 674 599564 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 75 7845 7860 ekkeekkdddddddkk 612 599590 2610 2626 Exon 18 GTTGTCGCAGCTGTTTT 88 7845 7861 eeekkdddddddkkeee 675 599565 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 65 7846 7861 ekkeekkdddddddkk 613 599591 2611 2627 Exon 18 TGTTGTCGCAGCTGTTT 94 7846 7862 eeekkdddddddkkeee 676 599566 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 72 7847 7862 ekkeekkdddddddkk 614 599592 2612 2628 Exon 18/ TTGTTGTCGCAGCTGTT 90 n/a n/a eeekkdddddddkkeee 677 Repeat 599567 2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 82 n/a n/a ekkeekkdddddddkk 615 Repeat 599593 2613 2629 Exon 18/ TTTGTTGTCGCAGCTGT 95 n/a n/a eeekkdddddddkkeee 678 Repeat 599568 2614 2629 Exon 18/ TTTGTTGTCGCAGCTG 92 n/a n/a ekkeekkdddddddkk 616 Repeat 599594 2614 2630 Exon 18/ TTTTGTTGTCGCAGCTG 86 n/a n/a eeekkdddddddkkeee 679 Repeat 599569 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 89 n/a n/a ekkeekkdddddddkk 617 Repeat 599595 2615 2631 Exon 18/ TTTTTGTTGTCGCAGCT 76 n/a n/a eeekkdddddddkkeee 680 Repeat 599570 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 95 n/a n/a ekkeekkdddddddkk 618 Repeat

Example 121: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) have nucleic acither and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE and (S)-cEt oligonucleotides, or as 5-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE, 6-7-6-MOE, 3-10-5 MOE, or 6-8-6 MOE gapmers.

The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification.

The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and five nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 142 Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ start stop Target inhi- start stop ID ISIS NO site site region Sequence bition site site Motif NO: 601152 2551 2566 Exon 18 AAACCCAAATCCTCAT 22 7786 7801 eekkddddddddkkee 557 601218 2551 2566 Exon 18 AAACCCAAATCCTCAT 21 7786 7801 ekkkddddddddkeee 557 601153 2552 2567 Exon 18 AAAACCCAAATCCTCA 27 7787 7802 eekkddddddddkkee 800 601219 2552 2567 Exon 18 AAAACCCAAATCCTCA 19 7787 7802 ekkkddddddddkeee 800 601154 2553 2568 Exon 18 GAAAACCCAAATCCTC 23 7788 7803 eekkddddddddkkee 558 601220 2553 2568 Exon 18 GAAAACCCAAATCCTC 24 7788 7803 ekkkddddddddkeee 558 601155 2554 2569 Exon 18 AGAAAACCCAAATCCT 20 7789 7804 eekkddddddddkkee 801 601221 2554 2569 Exon 18 AGAAAACCCAAATCCT 0 7789 7804 ekkkddddddddkeee 801 601156 2555 2570 Exon 18 TAGAAAACCCAAATCC 11 7790 7805 eekkddddddddkkee 559 601222 2555 2570 Exon 18 TAGAAAACCCAAATCC 23 7790 7805 ekkkddddddddkeee 559 601157 2556 2571 Exon 18 ATAGAAAACCCAAATC 9 7791 7806 eekkddddddddkkee 560 601223 2556 2571 Exon 18 ATAGAAAACCCAAATC 0 7791 7806 ekkkddddddddkeee 560 601158 2557 2572 Exon 18 TATAGAAAACCCAAAT 0 7792 7807 eekkddddddddkkee 802 601224 2557 2572 Exon 18 TATAGAAAACCCAAAT 0 7792 7807 ekkkddddddddkeee 802 601159 2558 2573 Exon 18 TTATAGAAAACCCAAA 2 7793 7808 eekkddddddddkkee 803 601225 2558 2573 Exon 18 TTATAGAAAACCCAAA 0 7793 7808 ekkkddddddddkeee 803 601160 2559 2574 Exon 18 CTTATAGAAAACCCAA 0 7794 7809 eekkddddddddkkee 561 601226 2559 2574 Exon 18 CTTATAGAAAACCCAA 0 7794 7809 ekkkddddddddkeee 561 601161 2560 2575 Exon 18 CCTTATAGAAAACCCA 1 7795 7810 eekkddddddddkkee 562 601227 2560 2575 Exon 18 CCTTATAGAAAACCCA 14 7795 7810 ekkkddddddddkeee 562 601162 2561 2576 Exon 18 CCCTTATAGAAAACCC 9 7796 7811 eekkddddddddkkee 563 601228 2561 2576 Exon 18 CCCTTATAGAAAACCC 9 7796 7811 ekkkddddddddkeee 563 601163 2562 2577 Exon 18 CCCCTTATAGAAAACC 0 7797 7812 eekkddddddddkkee 564 601164 2563 2578 Exon 18 ACCCCTTATAGAAAAC 3 7798 7813 eekkddddddddkkee 565 601165 2564 2579 Exon 18 AACCCCTTATAGAAAA 0 7799 7814 eekkddddddddkkee 566 601166 2565 2580 Exon 18 AAACCCCTTATAGAAA 0 7800 7815 eekkddddddddkkee 567 601167 2566 2581 Exon 18 GAAACCCCTTATAGAA 0 7801 7816 eekkddddddddkkee 568 601168 2567 2582 Exon 18 GGAAACCCCTTATAGA 0 7802 7817 eekkddddddddkkee 569 601169 2568 2583 Exon 18 AGGAAACCCCTTATAG 0 7803 7818 eekkddddddddkkee 570 601170 2569 2584 Exon 18 CAGGAAACCCCTTATA 10 7804 7819 eekkddddddddkkee 571 601171 2570 2585 Exon 18 GCAGGAAACCCCTTAT 9 7805 7820 eekkddddddddkkee 572 601172 2571 2586 Exon 18 AGCAGGAAACCCCTTA 15 7806 7821 eekkddddddddkkee 573 601173 2572 2587 Exon 18 CAGCAGGAAACCCCTT 29 7807 7822 eekkddddddddkkee 574 601174 2573 2588 Exon 18 CCAGCAGGAAACCCCT 25 7808 7823 eekkddddddddkkee 575 601175 2574 2589 Exon 18 TCCAGCAGGAAACCCC 15 7809 7824 eekkddddddddkkee 576 601176 2575 2590 Exon 18 GTCCAGCAGGAAACCC 18 7810 7825 eekkddddddddkkee 577 601177 2576 2591 Exon 18 TGTCCAGCAGGAAACC 10 7811 7826 eekkddddddddkkee 578 601178 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 11 7812 7827 eekkddddddddkkee 579 601179 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 19 7813 7828 eekkddddddddkkee 580 601180 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 7 7814 7829 eekkddddddddkkee 581 601181 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 3 7815 7830 eekkddddddddkkee 582 601182 2581 2596 Exon 18 GCCCCTGTCCAGCAGG 0 7816 7831 eekkddddddddkkee 583 601183 2582 2597 Exon 18 CGCCCCTGTCCAGCAG 4 7817 7832 eekkddddddddkkee 584 601184 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 14 7818 7833 eekkddddddddkkee 585 601185 2584 2599 Exon 18 CACGCCCCTGTCCAGC 26 7819 7834 eekkddddddddkkee 586 601186 2585 2600 Exon 18 CCACGCCCCTGTCCAG 8 7820 7835 eekkddddddddkkee 587 601187 2586 2601 Exon 18 CCCACGCCCCTGTCCA 18 7821 7836 eekkddddddddkkee 588 601188 2587 2602 Exon 18 TCCCACGCCCCTGTCC 20 7822 7837 eekkddddddddkkee 589 601189 2588 2603 Exon 18 ATCCCACGCCCCTGTC 12 7823 7838 eekkddddddddkkee 590 601190 2589 2604 Exon 18 AATCCCACGCCCCTGT 33 7824 7839 eekkddddddddkkee 591 601191 2590 2605 Exon 18 CAATCCCACGCCCCTG 52 7825 7840 eekkddddddddkkee 592 601192 2591 2606 Exon 18 TCAATCCCACGCCCCT 46 7826 7841 eekkddddddddkkee 593 601193 2592 2607 Exon 18 TTCAATCCCACGCCCC 30 7827 7842 eekkddddddddkkee 594 601194 2593 2608 Exon 18 ATTCAATCCCACGCCC 41 7828 7843 eekkddddddddkkee 595 601195 2594 2609 Exon 18 AATTCAATCCCACGCC 40 7829 7844 eekkddddddddkkee 596 601196 2595 2610 Exon 18 TAATTCAATCCCACGC 71 7830 7845 eekkddddddddkkee 597 601197 2596 2611 Exon 18 TTAATTCAATCCCACG 42 7831 7846 eekkddddddddkkee 598 601198 2597 2612 Exon 18 TTTAATTCAATCCCAC 63 7832 7847 eekkddddddddkkee 599 601199 2598 2613 Exon 18 TTTTAATTCAATCCCA 51 7833 7848 eekkddddddddkkee 600 601200 2599 2614 Exon 18 GTTTTAATTCAATCCC 65 7834 7849 eekkddddddddkkee 601 601201 2600 2615 Exon 18 TGTTTTAATTCAATCC 49 7835 7850 eekkddddddddkkee 602 601202 2601 2616 Exon 18 CTGTTTTAATTCAATC 33 7836 7851 eekkddddddddkkee 603 601203 2602 2617 Exon 18 GCTGTTTTAATTCAAT 63 7837 7852 eekkddddddddkkee 604 601204 2603 2618 Exon 18 AGCTGTTTTAATTCAA 69 7838 7853 eekkddddddddkkee 605 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATT 73 7839 7858 eeeeeddddddddddeeeee 317 CA 601205 2604 2619 Exon 18 CAGCTGTTTTAATTCA 51 7839 7854 eekkddddddddkkee 606 601206 2605 2620 Exon 18 GCAGCTGTTTTAATTC 43 7840 7855 eekkddddddddkkee 607 601207 2606 2621 Exon 18 CGCAGCTGTTTTAATT 52 7841 7856 eekkddddddddkkee 608 601208 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 61 7842 7857 eekkddddddddkkee 609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 75 7843 7858 eekddddddddddkke 610 601209 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 73 7843 7858 eekkddddddddkkee 610 601210 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 80 7844 7859 eekkddddddddkkee 611 601211 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 64 7845 7860 eekkddddddddkkee 612 601212 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 86 7846 7861 eekkddddddddkkee 613 601213 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 87 7847 7862 eekkddddddddkkee 614 601214 2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 84 n/a n/a eekkddddddddkkee 615 Repeat 601215 2614 2629 Exon 18/ TTTGTTGTCGCAGCTG 78 n/a n/a eekkddddddddkkee 616 Repeat 601216 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 73 n/a n/a eekkddddddddkkee 617 Repeat 601217 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 66 n/a n/a eekkddddddddkkee 618 Repeat

TABLE 143 Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 % NO: 2 NO: 2 SEQ start stop Target inhi- start stop ID ISIS NO site site region Sequence bition site site Motif NO: 601284 2551 2566 Exon 18 AAACCCAAATCCTCAT 8 7786 7801 ekkddddddddkkeee 557 601285 2552 2567 Exon 18 AAAACCCAAATCCTCA 15 7787 7802 ekkddddddddkkeee 800 601286 2553 2568 Exon 18 GAAAACCCAAATCCTC 21 7788 7803 ekkddddddddkkeee 558 601287 2554 2569 Exon 18 AGAAAACCCAAATCCT 9 7789 7804 ekkddddddddkkeee 801 601288 2555 2570 Exon 18 TAGAAAACCCAAATCC 0 7790 7805 ekkddddddddkkeee 559 601289 2556 2571 Exon 18 ATAGAAAACCCAAATC 40 7791 7806 ekkddddddddkkeee 560 601290 2557 2572 Exon 18 TATAGAAAACCCAAAT 16 7792 7807 ekkddddddddkkeee 802 601291 2558 2573 Exon 18 TTATAGAAAACCCAAA 15 7793 7808 ekkddddddddkkeee 803 601292 2559 2574 Exon 18 CTTATAGAAAACCCAA 5 7794 7809 ekkddddddddkkeee 561 601293 2560 2575 Exon 18 CCTTATAGAAAACCCA 15 7795 7810 ekkddddddddkkeee 562 601294 2561 2576 Exon 18 CCCTTATAGAAAACCC 3 7796 7811 ekkddddddddkkeee 563 601229 2562 2577 Exon 18 CCCCTTATAGAAAACC 15 7797 7812 ekkkddddddddkeee 564 601295 2562 2577 Exon 18 CCCCTTATAGAAAACC 5 7797 7812 ekkddddddddkkeee 564 601230 2563 2578 Exon 18 ACCCCTTATAGAAAAC 14 7798 7813 ekkkddddddddkeee 565 601296 2563 2578 Exon 18 ACCCCTTATAGAAAAC 0 7798 7813 ekkddddddddkkeee 565 601231 2564 2579 Exon 18 AACCCCTTATAGAAAA 14 7799 7814 ekkkddddddddkeee 566 601297 2564 2579 Exon 18 AACCCCTTATAGAAAA 14 7799 7814 ekkddddddddkkeee 566 601232 2565 2580 Exon 18 AAACCCCTTATAGAAA 15 7800 7815 ekkkddddddddkeee 567 601298 2565 2580 Exon 18 AAACCCCTTATAGAAA 7 7800 7815 ekkddddddddkkeee 567 601233 2566 2581 Exon 18 GAAACCCCTTATAGAA 0 7801 7816 ekkkddddddddkeee 568 601299 2566 2581 Exon 18 GAAACCCCTTATAGAA 0 7801 7816 ekkddddddddkkeee 568 601234 2567 2582 Exon 18 GGAAACCCCTTATAGA 0 7802 7817 ekkkddddddddkeee 569 601300 2567 2582 Exon 18 GGAAACCCCTTATAGA 9 7802 7817 ekkddddddddkkeee 569 601235 2568 2583 Exon 18 AGGAAACCCCTTATAG 3 7803 7818 ekkkddddddddkeee 570 601301 2568 2583 Exon 18 AGGAAACCCCTTATAG 14 7803 7818 ekkddddddddkkeee 570 601236 2569 2584 Exon 18 CAGGAAACCCCTTATA 0 7804 7819 ekkkddddddddkeee 571 601302 2569 2584 Exon 18 CAGGAAACCCCTTATA 0 7804 7819 ekkddddddddkkeee 571 601237 2570 2585 Exon 18 GCAGGAAACCCCTTAT 16 7805 7820 ekkkddddddddkeee 572 601303 2570 2585 Exon 18 GCAGGAAACCCCTTAT 16 7805 7820 ekkddddddddkkeee 572 601238 2571 2586 Exon 18 AGCAGGAAACCCCTTA 11 7806 7821 ekkkddddddddkeee 573 601304 2571 2586 Exon 18 AGCAGGAAACCCCTTA 10 7806 7821 ekkddddddddkkeee 573 601239 2572 2587 Exon 18 CAGCAGGAAACCCCTT 21 7807 7822 ekkkddddddddkeee 574 601305 2572 2587 Exon 18 CAGCAGGAAACCCCTT 7 7807 7822 ekkddddddddkkeee 574 601240 2573 2588 Exon 18 CCAGCAGGAAACCCCT 6 7808 7823 ekkkddddddddkeee 575 601241 2574 2589 Exon 18 TCCAGCAGGAAACCCC 10 7809 7824 ekkkddddddddkeee 576 601242 2575 2590 Exon 18 GTCCAGCAGGAAACCC 19 7810 7825 ekkkddddddddkeee 577 601243 2576 2591 Exon 18 TGTCCAGCAGGAAACC 10 7811 7826 ekkkddddddddkeee 578 601244 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 28 7812 7827 ekkkddddddddkeee 579 601245 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 5 7813 7828 ekkkddddddddkeee 580 601246 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 18 7814 7829 ekkkddddddddkeee 581 601247 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 4 7815 7830 ekkkddddddddkeee 582 601248 2581 2596 Exon 18 GCCCCTGTCCAGCAGG 6 7816 7831 ekkkddddddddkeee 583 601249 2582 2597 Exon 18 CGCCCCTGTCCAGCAG 18 7817 7832 ekkkddddddddkeee 584 601250 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 26 7818 7833 ekkkddddddddkeee 585 601251 2584 2599 Exon 18 CACGCCCCTGTCCAGC 27 7819 7834 ekkkddddddddkeee 586 601252 2585 2600 Exon 18 CCACGCCCCTGTCCAG 21 7820 7835 ekkkddddddddkeee 587 601253 2586 2601 Exon 18 CCCACGCCCCTGTCCA 0 7821 7836 ekkkddddddddkeee 588 601254 2587 2602 Exon 18 TCCCACGCCCCTGTCC 31 7822 7837 ekkkddddddddkeee 589 601255 2588 2603 Exon 18 ATCCCACGCCCCTGTC 3 7823 7838 ekkkddddddddkeee 590 601256 2589 2604 Exon 18 AATCCCACGCCCCTGT 21 7824 7839 ekkkddddddddkeee 591 601257 2590 2605 Exon 18 CAATCCCACGCCCCTG 47 7825 7840 ekkkddddddddkeee 592 601258 2591 2606 Exon 18 TCAATCCCACGCCCCT 48 7826 7841 ekkkddddddddkeee 593 601259 2592 2607 Exon 18 TTCAATCCCACGCCCC 38 7827 7842 ekkkddddddddkeee 594 601260 2593 2608 Exon 18 ATTCAATCCCACGCCC 33 7828 7843 ekkkddddddddkeee 595 601261 2594 2609 Exon 18 AATTCAATCCCACGCC 17 7829 7844 ekkkddddddddkeee 596 601262 2595 2610 Exon 18 TAATTCAATCCCACGC 40 7830 7845 ekkkddddddddkeee 597 601263 2596 2611 Exon 18 TTAATTCAATCCCACG 31 7831 7846 ekkkddddddddkeee 598 601264 2597 2612 Exon 18 TTTAATTCAATCCCAC 72 7832 7847 ekkkddddddddkeee 599 601265 2598 2613 Exon 18 TTTTAATTCAATCCCA 48 7833 7848 ekkkddddddddkeee 600 601266 2599 2614 Exon 18 GTTTTAATTCAATCCC 64 7834 7849 ekkkddddddddkeee 601 601267 2600 2615 Exon 18 TGTTTTAATTCAATCC 43 7835 7850 ekkkddddddddkeee 602 601268 2601 2616 Exon 18 CTGTTTTAATTCAATC 44 7836 7851 ekkkddddddddkeee 603 601269 2602 2617 Exon 18 GCTGTTTTAATTCAAT 66 7837 7852 ekkkddddddddkeee 604 601270 2603 2618 Exon 18 AGCTGTTTTAATTCAA 47 7838 7853 ekkkddddddddkeee 605 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATT 3 7839 7858 eeeeeddddddddddeeeee 317 CA 601271 2604 2619 Exon 18 CAGCTGTTTTAATTCA 26 7839 7854 ekkkddddddddkeee 606 601272 2605 2620 Exon 18 GCAGCTGTTTTAATTC 33 7840 7855 ekkkddddddddkeee 607 601273 2606 2621 Exon 18 CGCAGCTGTTTTAATT 34 7841 7856 ekkkddddddddkeee 608 601274 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 39 7842 7857 ekkkddddddddkeee 609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 72 7843 7858 eekddddddddddkke 610 601275 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 65 7843 7858 ekkkddddddddkeee 610 601276 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 65 7844 7859 ekkkddddddddkeee 611 601277 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 51 7845 7860 ekkkddddddddkeee 612 601278 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 78 7846 7861 ekkkddddddddkeee 613 601279 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 79 7847 7862 ekkkddddddddkeee 614 601280 2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 70 n/a n/a ekkkddddddddkeee 615 Repeat 601281 2614 2629 Exon 18/ TTTGTTGTCGCAGCTG 78 n/a n/a ekkkddddddddkeee 616 Repeat 601282 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 68 n/a n/a ekkkddddddddkeee 617 Repeat 601283 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 61 n/a n/a ekkkddddddddkeee 618 Repeat

TABLE 144 Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ start stop Target % start stop ID ISIS NO site site region Sequence inhibition site site Motif NO: 601306 2573 2588 Exon 18 CCAGCAGGAAACCCCT 22 7808 7823 ekkddddddddkkeee 575 601307 2574 2589 Exon 18 TCCAGCAGGAAACCCC 22 7809 7824 ekkddddddddkkeee 576 601308 2575 2590 Exon 18 GTCCAGCAGGAAACCC 33 7810 7825 ekkddddddddkkeee 577 601309 2576 2591 Exon 18 TGTCCAGCAGGAAACC 33 7811 7826 ekkddddddddkkeee 578 601310 2577 2592 Exon 18 CTGTCCAGCAGGAAAC 28 7812 7827 ekkddddddddkkeee 579 601311 2578 2593 Exon 18 CCTGTCCAGCAGGAAA 33 7813 7828 ekkddddddddkkeee 580 601312 2579 2594 Exon 18 CCCTGTCCAGCAGGAA 13 7814 7829 ekkddddddddkkeee 581 601313 2580 2595 Exon 18 CCCCTGTCCAGCAGGA 32 7815 7830 ekkddddddddkkeee 582 601314 2581 2596 Exon 18 GCCCCTGTCCAGCAGG 0 7816 7831 ekkddddddddkkeee 583 601315 2582 2597 Exon 18 CGCCCCTGTCCAGCAG 36 7817 7832 ekkddddddddkkeee 584 601316 2583 2598 Exon 18 ACGCCCCTGTCCAGCA 39 7818 7833 ekkddddddddkkeee 585 601317 2584 2599 Exon 18 CACGCCCCTGTCCAGC 33 7819 7834 ekkddddddddkkeee 586 601356 2584 2599 Exon 18 CACGCCCCTGTCCAGC 27 7819 7834 kkkddddddddkeeee 586 601318 2585 2600 Exon 18 CCACGCCCCTGTCCAG 35 7820 7835 ekkddddddddkkeee 587 601357 2585 2600 Exon 18 CCACGCCCCTGTCCAG 26 7820 7835 kkkddddddddkeeee 587 601319 2586 2601 Exon 18 CCCACGCCCCTGTCCA 33 7821 7836 ekkddddddddkkeee 588 601358 2586 2601 Exon 18 CCCACGCCCCTGTCCA 26 7821 7836 kkkddddddddkeeee 588 601320 2587 2602 Exon 18 TCCCACGCCCCTGTCC 25 7822 7837 ekkddddddddkkeee 589 601359 2587 2602 Exon 18 TCCCACGCCCCTGTCC 23 7822 7837 kkkddddddddkeeee 589 601321 2588 2603 Exon 18 ATCCCACGCCCCTGTC 50 7823 7838 ekkddddddddkkeee 590 601360 2588 2603 Exon 18 ATCCCACGCCCCTGTC 33 7823 7838 kkkddddddddkeeee 590 601322 2589 2604 Exon 18 AATCCCACGCCCCTGT 52 7824 7839 ekkddddddddkkeee 591 601361 2589 2604 Exon 18 AATCCCACGCCCCTGT 48 7824 7839 kkkddddddddkeeee 591 601323 2590 2605 Exon 18 CAATCCCACGCCCCTG 67 7825 7840 ekkddddddddkkeee 592 601362 2590 2605 Exon 18 CAATCCCACGCCCCTG 51 7825 7840 kkkddddddddkeeee 592 601324 2591 2606 Exon 18 TCAATCCCACGCCCCT 42 7826 7841 ekkddddddddkkeee 593 601363 2591 2606 Exon 18 TCAATCCCACGCCCCT 42 7826 7841 kkkddddddddkeeee 593 601325 2592 2607 Exon 18 TTCAATCCCACGCCCC 52 7827 7842 ekkddddddddkkeee 594 601364 2592 2607 Exon 18 TTCAATCCCACGCCCC 48 7827 7842 kkkddddddddkeeee 594 601326 2593 2608 Exon 18 ATTCAATCCCACGCCC 27 7828 7843 ekkddddddddkkeee 595 601365 2593 2608 Exon 18 ATTCAATCCCACGCCC 36 7828 7843 kkkddddddddkeeee 595 601327 2594 2609 Exon 18 AATTCAATCCCACGCC 66 7829 7844 ekkddddddddkkeee 596 601366 2594 2609 Exon 18 AATTCAATCCCACGCC 49 7829 7844 kkkddddddddkeeee 596 601328 2595 2610 Exon 18 TAATTCAATCCCACGC 55 7830 7845 ekkddddddddkkeee 597 601367 2595 2610 Exon 18 TAATTCAATCCCACGC 57 7830 7845 kkkddddddddkeeee 597 601329 2596 2611 Exon 18 TTAATTCAATCCCACG 69 7831 7846 ekkddddddddkkeee 598 601368 2596 2611 Exon 18 TTAATTCAATCCCACG 68 7831 7846 kkkddddddddkeeee 598 601330 2597 2612 Exon 18 TTTAATTCAATCCCAC 58 7832 7847 ekkddddddddkkeee 599 601369 2597 2612 Exon 18 TTTAATTCAATCCCAC 65 7832 7847 kkkddddddddkeeee 599 601331 2598 2613 Exon 18 TTTTAATTCAATCCCA 45 7833 7848 ekkddddddddkkeee 600 601370 2598 2613 Exon 18 TTTTAATTCAATCCCA 42 7833 7848 kkkddddddddkeeee 600 601332 2599 2614 Exon 18 GTTTTAATTCAATCCC 84 7834 7849 ekkddddddddkkeee 601 601371 2599 2614 Exon 18 GTTTTAATTCAATCCC 79 7834 7849 kkkddddddddkeeee 601 601333 2600 2615 Exon 18 TGTTTTAATTCAATCC 61 7835 7850 ekkddddddddkkeee 602 601372 2600 2615 Exon 18 TGTTTTAATTCAATCC 71 7835 7850 kkkddddddddkeeee 602 601334 2601 2616 Exon 18 CTGTTTTAATTCAATC 61 7836 7851 ekkddddddddkkeee 603 601373 2601 2616 Exon 18 CTGTTTTAATTCAATC 57 7836 7851 kkkddddddddkeeee 603 601335 2602 2617 Exon 18 GCTGTTTTAATTCAAT 73 7837 7852 ekkddddddddkkeee 604 601374 2602 2617 Exon 18 GCTGTTTTAATTCAAT 66 7837 7852 kkkddddddddkeeee 604 601336 2603 2618 Exon 18 AGCTGTTTTAATTCAA 64 7838 7853 ekkddddddddkkeee 605 601375 2603 2618 Exon 18 AGCTGTTTTAATTCAA 61 7838 7853 kkkddddddddkeeee 605 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATT 66 7839 7858 eeeeedddddddddde 317 CA eeee 601337 2604 2619 Exon 18 CAGCTGTTTTAATTCA 53 7839 7854 ekkddddddddkkeee 606 601376 2604 2619 Exon 18 CAGCTGTTTTAATTCA 39 7839 7854 kkkddddddddkeeee 606 601338 2605 2620 Exon 18 GCAGCTGTTTTAATTC 67 7840 7855 ekkddddddddkkeee 607 601377 2605 2620 Exon 18 GCAGCTGTTTTAATTC 67 7840 7855 kkkddddddddkeeee 607 601339 2606 2621 Exon 18 CGCAGCTGTTTTAATT 63 7841 7856 ekkddddddddkkeee 608 601378 2606 2621 Exon 18 CGCAGCTGTTTTAATT 60 7841 7856 kkkddddddddkeeee 608 601340 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 40 7842 7857 ekkddddddddkkeee 609 601379 2607 2622 Exon 18 TCGCAGCTGTTTTAAT 36 7842 7857 kkkddddddddkeeee 609 588860 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 84 7843 7858 eekddddddddddkke 610 601341 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 74 7843 7858 ekkddddddddkkeee 610 601380 2608 2623 Exon 18 GTCGCAGCTGTTTTAA 78 7843 7858 kkkddddddddkeeee 610 601342 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 68 7844 7859 ekkddddddddkkeee 611 601381 2609 2624 Exon 18 TGTCGCAGCTGTTTTA 66 7844 7859 kkkddddddddkeeee 611 601343 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 71 7845 7860 ekkddddddddkkeee 612 601382 2610 2625 Exon 18 TTGTCGCAGCTGTTTT 84 7845 7860 kkkddddddddkeeee 612 601344 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 87 7846 7861 ekkddddddddkkeee 613 601383 2611 2626 Exon 18 GTTGTCGCAGCTGTTT 85 7846 7861 kkkddddddddkeeee 613 601345 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 82 7847 7862 ekkddddddddkkeee 614 601384 2612 2627 Exon 18 TGTTGTCGCAGCTGTT 79 7847 7862 kkkddddddddkeeee 614 601346 2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 73 n/a n/a ekkddddddddkkeee 615 Repeat 601385 2613 2628 Exon 18/ TTGTTGTCGCAGCTGT 84 n/a n/a kkkddddddddkeeee 615 Repeat 601347 2614 2629 Exon 18/ TTTGTTGTCGCAGCTG 70 n/a n/a ekkddddddddkkeee 616 Repeat 601386 2614 2629 Exon 18/ TTTGTTGTCGCAGCTG 71 n/a n/a kkkddddddddkeeee 616 Repeat 601348 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 71 n/a n/a ekkddddddddkkeee 617 Repeat 601387 2615 2630 Exon 18/ TTTTGTTGTCGCAGCT 76 n/a n/a kkkddddddddkeeee 617 Repeat 601349 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 71 n/a n/a ekkddddddddkkeee 618 Repeat 601388 2616 2631 Exon 18/ TTTTTGTTGTCGCAGC 67 n/a n/a kkkddddddddkeeee 618 Repeat

TABLE 145 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: SEQ ISIS start stop Target % start 2 stop ID NO site site region Sequence inhibition site site Motif NO: 599357 2582 2600 Exon 18 CCACGCCCCTGTCCAGCAG 26 7817 7835 5-9-5 708 599358 2583 2601 Exon 18 CCCACGCCCCTGTCCAGCA 22 7818 7836 5-9-5 709 599359 2584 2602 Exon 18 TCCCACGCCCCTGTCCAGC 13 7819 7837 5-9-5 710 599360 2585 2603 Exon 18 ATCCCACGCCCCTGTCCAG 7 7820 7838 5-9-5 711 599361 2586 2604 Exon 18 AATCCCACGCCCCTGTCCA 11 7821 7839 5-9-5 712 599362 2587 2605 Exon 18 CAATCCCACGCCCCTGTCC 14 7822 7840 5-9-5 713 599363 2588 2606 Exon 18 TCAATCCCACGCCCCTGTC 17 7823 7841 5-9-5 714 599364 2589 2607 Exon 18 TTCAATCCCACGCCCCTGT 20 7824 7842 5-9-5 715 599365 2590 2608 Exon 18 ATTCAATCCCACGCCCCTG 22 7825 7843 5-9-5 716 599366 2591 2609 Exon 18 AATTCAATCCCACGCCCCT 13 7826 7844 5-9-5 717 599367 2592 2610 Exon 18 TAATTCAATCCCACGCCCC 11 7827 7845 5-9-5 718 599368 2593 2611 Exon 18 TTAATTCAATCCCACGCCC 10 7828 7846 5-9-5 719 599369 2594 2612 Exon 18 TTTAATTCAATCCCACGCC 19 7829 7847 5-9-5 720 599370 2595 2613 Exon 18 TTTTAATTCAATCCCACGC 23 7830 7848 5-9-5 721 599371 2596 2614 Exon 18 GTTTTAATTCAATCCCACG 4 7831 7849 5-9-5 722 599372 2597 2615 Exon 18 TGTTTTAATTCAATCCCAC 16 7832 7850 5-9-5 723 599373 2598 2616 Exon 18 CTGTTTTAATTCAATCCCA 3 7833 7851 5-9-5 724 599374 2599 2617 Exon 18 GCTGTTTTAATTCAATCCC 10 7834 7852 5-9-5 725 599375 2600 2618 Exon 18 AGCTGTTTTAATTCAATCC 17 7835 7853 5-9-5 726 599376 2601 2619 Exon 18 CAGCTGTTTTAATTCAATC 18 7836 7854 5-9-5 727 599377 2602 2620 Exon 18 GCAGCTGTTTTAATTCAAT 22 7837 7855 5-9-5 728 599378 2603 2621 Exon 18 CGCAGCTGTTTTAATTCAA 11 7838 7856 5-9-5 729 599511 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 7 7787 7806 6-8-6 410 599389 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 22 7788 7807 6-8-6 411 599390 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 21 7789 7808 6-8-6 412 599391 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 27 7790 7809 6-8-6 413 599392 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC 30 7791 7810 6-8-6 414 599393 2557 2576 Exon 18 CCCTTATAGAAAACCCAAAT 30 7792 7811 6-8-6 415 599394 2558 2577 Exon 18 CCCCTTATAGAAAACCCAAA 28 7793 7812 6-8-6 416 599395 2559 2578 Exon 18 ACCCCTTATAGAAAACCCAA 23 7794 7813 6-8-6 417 599396 2560 2579 Exon 18 AACCCCTTATAGAAAACCCA 53 7795 7814 6-8-6 418 599397 2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 33 7796 7815 6-8-6 419 599398 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 58 7797 7816 6-8-6 420 599399 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 23 7798 7817 6-8-6 421 599400 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 54 7799 7818 6-8-6 422 599401 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 30 7800 7819 6-8-6 423 599402 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA 25 7801 7820 6-8-6 424 599403 2567 2586 Exon 18 AGCAGGAAACCCCTTATAGA 17 7802 7821 6-8-6 425 599404 2568 2587 Exon 18 CAGCAGGAAACCCCTTATAG 20 7803 7822 6-8-6 426 599405 2569 2588 Exon 18 CCAGCAGGAAACCCCTTATA 12 7804 7823 6-8-6 427 599406 2570 2589 Exon 18 TCCAGCAGGAAACCCCTTAT 51 7805 7824 6-8-6 428 599407 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 39 7806 7825 6-8-6 237 599408 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 53 7807 7826 6-8-6 429 599409 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 65 7808 7827 6-8-6 430 599410 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 56 7809 7828 6-8-6 431 599411 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 60 7810 7829 6-8-6 432 599412 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC 61 7811 7830 6-8-6 433 599413 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 40 7812 7831 6-8-6 238 599414 2578 2597 Exon 18 CGCCCCTGTCCAGCAGGAAA 41 7813 7832 6-8-6 434 599415 2579 2598 Exon 18 ACGCCCCTGTCCAGCAGGAA 37 7814 7833 6-8-6 435 599416 2580 2599 Exon 18 CACGCCCCTGTCCAGCAGGA 54 7815 7834 6-8-6 436 599417 2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 36 7816 7835 6-8-6 437 599418 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 53 7817 7836 6-8-6 438 599419 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 54 7818 7837 6-8-6 439 599420 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 50 7819 7838 6-8-6 440 599421 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 48 7820 7839 6-8-6 441 599422 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA 55 7821 7840 6-8-6 442 599423 2587 2606 Exon 18 TCAATCCCACGCCCCTGTCC 75 7822 7841 6-8-6 443 599424 2588 2607 Exon 18 TTCAATCCCACGCCCCTGTC 69 7823 7842 6-8-6 444 599425 2589 2608 Exon 18 ATTCAATCCCACGCCCCTGT 77 7824 7843 6-8-6 445 599426 2590 2609 Exon 18 AATTCAATCCCACGCCCCTG 60 7825 7844 6-8-6 446 599427 2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 72 7826 7845 6-8-6 447 599428 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 81 7827 7846 6-8-6 448 599429 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 68 7828 7847 6-8-6 449 599430 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 58 7829 7848 6-8-6 450 599431 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 70 7830 7849 6-8-6 451 599432 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG 85 7831 7850 6-8-6 452 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 85 7839 7858 5-10-5 317 599379 2604 2622 Exon 18 TCGCAGCTGTTTTAATTCA 73 7839 7857 5-9-5 730 599380 2605 2623 Exon 18 GTCGCAGCTGTTTTAATTC 77 7840 7858 5-9-5 731 599381 2606 2624 Exon 18 TGTCGCAGCTGTTTTAATT 69 7841 7859 5-9-5 732 599382 2607 2625 Exon 18 TTGTCGCAGCTGTTTTAAT 58 7842 7860 5-9-5 733 599383 2608 2626 Exon 18 GTTGTCGCAGCTGTTTTAA 52 7843 7861 5-9-5 734 599384 2609 2627 Exon 18 TGTTGTCGCAGCTGTTTTA 63 7844 7862 5-9-5 735 599385 2610 2628 Exon 18/ TTGTTGTCGCAGCTGTTTT 53 n/a n/a 5-9-5 736 Repeat 599386 2611 2629 Exon 18/ TTTGTTGTCGCAGCTGTTT 63 n/a n/a 5-9-5 737 Repeat 599387 2612 2630 Exon 18/ TTTTGTTGTCGCAGCTGTT 64 n/a n/a 5-9-5 438 Repeat 599388 2613 2631 Exon 18/ TTTTTGTTGTCGCAGCTGT 66 n/a n/a 5-9-5 739 Repeat

TABLE 146 Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ ID ID SEQ SEQ NO: 1 NO: 1 ID NO: ID NO: SEQ ISIS start stop Target % 2 start 2 stop ID NO site site region Sequence inhibition site site Motif NO: 599213 2553 2570 Exon 18 TAGAAAACCCAAATCCTC 0 7788 7805 3-10-5 785 599214 2554 2571 Exon 18 ATAGAAAACCCAAATCCT 0 7789 7806 3-10-5 786 599215 2555 2572 Exon 18 TATAGAAAACCCAAATCC 36 7790 7807 3-10-5 787 599216 2556 2573 Exon 18 TTATAGAAAACCCAAATC 8 7791 7808 3-10-5 788 599217 2557 2574 Exon 18 CTTATAGAAAACCCAAAT 5 7792 7809 3-10-5 789 599218 2558 2575 Exon 18 CCTTATAGAAAACCCAAA 0 7793 7810 3-10-5 790 599219 2559 2576 Exon 18 CCCTTATAGAAAACCCAA 8 7794 7811 3-10-5 791 599220 2560 2577 Exon 18 CCCCTTATAGAAAACCCA 0 7795 7812 3-10-5 740 599221 2561 2578 Exon 18 ACCCCTTATAGAAAACCC 54 7796 7813 3-10-5 741 599222 2562 2579 Exon 18 AACCCCTTATAGAAAACC 3 7797 7814 3-10-5 742 599223 2563 2580 Exon 18 AAACCCCTTATAGAAAAC 0 7798 7815 3-10-5 743 599224 2564 2581 Exon 18 GAAACCCCTTATAGAAAA 0 7799 7816 3-10-5 744 599225 2566 2583 Exon 18 AGGAAACCCCTTATAGAA 60 7801 7818 3-10-5 745 599226 2567 2584 Exon 18 CAGGAAACCCCTTATAGA 0 7802 7819 3-10-5 746 599227 2568 2585 Exon 18 GCAGGAAACCCCTTATAG 37 7803 7820 3-10-5 747 599228 2569 2586 Exon 18 AGCAGGAAACCCCTTATA 0 7804 7821 3-10-5 748 599229 2570 2587 Exon 18 CAGCAGGAAACCCCTTAT 39 7805 7822 3-10-5 749 599230 2571 2588 Exon 18 CCAGCAGGAAACCCCTTA 10 7806 7823 3-10-5 750 599231 2572 2589 Exon 18 TCCAGCAGGAAACCCCTT 16 7807 7824 3-10-5 751 599232 2573 2590 Exon 18 GTCCAGCAGGAAACCCCT 9 7808 7825 3-10-5 752 599233 2574 2591 Exon 18 TGTCCAGCAGGAAACCCC 44 7809 7826 3-10-5 753 599234 2575 2592 Exon 18 CTGTCCAGCAGGAAACCC 14 7810 7827 3-10-5 754 599235 2576 2593 Exon 18 CCTGTCCAGCAGGAAACC 0 7811 7828 3-10-5 755 599236 2577 2594 Exon 18 CCCTGTCCAGCAGGAAAC 43 7812 7829 3-10-5 756 599237 2578 2595 Exon 18 CCCCTGTCCAGCAGGAAA 0 7813 7830 3-10-5 757 599238 2580 2597 Exon 18 CGCCCCTGTCCAGCAGGA 9 7815 7832 3-10-5 758 599239 2581 2598 Exon 18 ACGCCCCTGTCCAGCAGG 36 7816 7833 3-10-5 759 599240 2582 2599 Exon 18 CACGCCCCTGTCCAGCAG 11 7817 7834 3-10-5 760 599241 2583 2600 Exon 18 CCACGCCCCTGTCCAGCA 51 7818 7835 3-10-5 761 599242 2584 2601 Exon 18 CCCACGCCCCTGTCCAGC 7 7819 7836 3-10-5 762 599243 2585 2602 Exon 18 TCCCACGCCCCTGTCCAG 47 7820 7837 3-10-5 763 599244 2586 2603 Exon 18 ATCCCACGCCCCTGTCCA 37 7821 7838 3-10-5 764 599245 2587 2604 Exon 18 AATCCCACGCCCCTGTCC 35 7822 7839 3-10-5 765 599246 2588 2605 Exon 18 CAATCCCACGCCCCTGTC 21 7823 7840 3-10-5 766 599247 2589 2606 Exon 18 TCAATCCCACGCCCCTGT 61 7824 7841 3-10-5 767 599248 2590 2607 Exon 18 TTCAATCCCACGCCCCTG 51 7825 7842 3-10-5 768 599249 2591 2608 Exon 18 ATTCAATCCCACGCCCCT 58 7826 7843 3-10-5 769 599250 2592 2609 Exon 18 AATTCAATCCCACGCCCC 49 7827 7844 3-10-5 770 599251 2593 2610 Exon 18 TAATTCAATCCCACGCCC 46 7828 7845 3-10-5 771 599252 2594 2611 Exon 18 TTAATTCAATCCCACGCC 32 7829 7846 3-10-5 772 599253 2595 2612 Exon 18 TTTAATTCAATCCCACGC 23 7830 7847 3-10-5 773 599254 2596 2613 Exon 18 TTTTAATTCAATCCCACG 0 7831 7848 3-10-5 774 599255 2597 2614 Exon 18 GTTTTAATTCAATCCCAC 61 7832 7849 3-10-5 775 599256 2598 2615 Exon 18 TGTTTTAATTCAATCCCA 64 7833 7850 3-10-5 776 599257 2599 2616 Exon 18 CTGTTTTAATTCAATCCC 66 7834 7851 3-10-5 777 599258 2600 2617 Exon 18 GCTGTTTTAATTCAATCC 59 7835 7852 3-10-5 778 599259 2601 2618 Exon 18 AGCTGTTTTAATTCAATC 40 7836 7853 3-10-5 779 599260 2602 2619 Exon 18 CAGCTGTTTTAATTCAAT 38 7837 7854 3-10-5 780 599261 2603 2620 Exon 18 GCAGCTGTTTTAATTCAA 54 7838 7855 3-10-5 781 599509 2552 2570 Exon 18 TAGAAAACCCAAATCCTCA 54 7787 7805 6-7-6 681 599273 2553 2571 Exon 18 ATAGAAAACCCAAATCCTC 0 7788 7806 6-7-6 682 599274 2554 2572 Exon 18 TATAGAAAACCCAAATCCT 57 7789 7807 6-7-6 683 599275 2556 2574 Exon 18 CTTATAGAAAACCCAAATC 0 7791 7809 6-7-6 684 599276 2557 2575 Exon 18 CCTTATAGAAAACCCAAAT 44 7792 7810 6-7-6 685 599277 2558 2576 Exon 18 CCCTTATAGAAAACCCAAA 0 7793 7811 6-7-6 686 599278 2559 2577 Exon 18 CCCCTTATAGAAAACCCAA 0 7794 7812 6-7-6 687 599279 2560 2578 Exon 18 ACCCCTTATAGAAAACCCA 20 7795 7813 6-7-6 688 599280 2561 2579 Exon 18 AACCCCTTATAGAAAACCC 70 7796 7814 6-7-6 689 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 85 7839 7858 5-10-5 317 599262 2604 2621 Exon 18 CGCAGCTGTTTTAATTCA 49 7839 7856 3-10-5 782 599263 2605 2622 Exon 18 TCGCAGCTGTTTTAATTC 49 7840 7857 3-10-5 783 599264 2606 2623 Exon 18 GTCGCAGCTGTTTTAATT 62 7841 7858 3-10-5 784 599265 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 63 7842 7859 3-10-5 792 599266 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 41 7843 7860 3-10-5 793 599267 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 52 7844 7861 3-10-5 794 599268 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 51 7845 7862 3-10-5 795 599269 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 58 n/a n/a 3-10-5 796 Repeat 599270 2612 2629 Exon 18/ TTTGTTGTCGCAGCTGTT 69 n/a n/a 3-10-5 797 Repeat 599271 2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 69 n/a n/a 3-10-5 798 Repeat 599272 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 72 n/a n/a 3-10-5 799 Repeat 599205 2607 2624 Exon 18 TGTCGCAGCTGTTTTAAT 54 7842 7859 5-8-5 792 599206 2608 2625 Exon 18 TTGTCGCAGCTGTTTTAA 62 7843 7860 5-8-5 793 599207 2609 2626 Exon 18 GTTGTCGCAGCTGTTTTA 62 7844 7861 5-8-5 794 599208 2610 2627 Exon 18 TGTTGTCGCAGCTGTTTT 66 7845 7862 5-8-5 795 599209 2611 2628 Exon 18/ TTGTTGTCGCAGCTGTTT 60 n/a n/a 5-8-5 796 Repeat 599210 2612 2629 Exon 18/ TTTGTTGTCGCAGCTGTT 62 n/a n/a 5-8-5 797 Repeat 599211 2613 2630 Exon 18/ TTTTGTTGTCGCAGCTGT 65 n/a n/a 5-8-5 798 Repeat 599212 2614 2631 Exon 18/ TTTTTGTTGTCGCAGCTG 67 n/a n/a 5-8-5 799 Repeat

TABLE 147 Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 or SEQ ID NO: 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS start stop Target % start stop ID NO site site region Sequence inhibition site site NO: 588570 150 169 Exon 1 TGGTCACATTCCCTTCCCCT 72 1871 1890 396 588571 152 171 Exon 1 CCTGGTCACATTCCCTTCCC 80 1873 1892 397 532614 154 173 Exon 1 GACCTGGTCACATTCCCTTC 65 1875 1894 12 588572 156 175 Exon 1 TAGACCTGGTCACATTCCCT 74 1877 1896 398 588573 158 177 Exon 1 CCTAGACCTGGTCACATTCC 72 1879 1898 399 588566 2189 2208 Exon 15 CCTTCCGAGTCAGCTTTTTC 66 6977 6996 400 588567 2191 2210 Exon 15 CTCCTTCCGAGTCAGCTTTT 66 6979 6998 401 532770 2193 2212 Exon 15 ACCTCCTTCCGAGTCAGCTT 64 6981 7000 198 588568 2195 2214 Exon 15 AGACCTCCTTCCGAGTCAGC 78 6983 7002 402 588569 2197 2216 Exon 15 GTAGACCTCCTTCCGAGTCA 74 6985 7004 403 588574 2453 2472 Exon 18 TTTGCCGCTTCTGGTTTTTG 71 7688 7707 404 588575 2455 2474 Exon 18 CTTTTGCCGCTTCTGGTTTT 72 7690 7709 405 532800 2457 2476 Exon 18 TGCTTTTGCCGCTTCTGGTT 71 7692 7711 228 588576 2459 2478 Exon 18 CCTGCTTTTGCCGCTTCTGG 59 7694 7713 406 588577 2461 2480 Exon 18 TACCTGCTTTTGCCGCTTCT 76 7696 7715 407 516350 2550 2569 Exon 18 AGAAAACCCAAATCCTCATC 58 7785 7804 408 588509 2551 2570 Exon 18 TAGAAAACCCAAATCCTCAT 6 7786 7805 409 588510 2552 2571 Exon 18 ATAGAAAACCCAAATCCTCA 10 7787 7806 410 588511 2553 2572 Exon 18 TATAGAAAACCCAAATCCTC 9 7788 7807 411 588512 2554 2573 Exon 18 TTATAGAAAACCCAAATCCT 80 7789 7808 412 588513 2555 2574 Exon 18 CTTATAGAAAACCCAAATCC 70 7790 7809 413 588514 2556 2575 Exon 18 CCTTATAGAAAACCCAAATC 71 7791 7810 414 588515 2557 2576 Exon 18 CCCTTATAGAAAACCCAAAT 78 7792 7811 415 588516 2558 2577 Exon 18 CCCCTTATAGAAAACCCAAA 72 7793 7812 416 588517 2559 2578 Exon 18 ACCCCTTATAGAAAACCCAA 80 7794 7813 417 588518 2560 2579 Exon 18 AACCCCTTATAGAAAACCCA 80 7795 7814 418 588519 2561 2580 Exon 18 AAACCCCTTATAGAAAACCC 62 7796 7815 419 588520 2562 2581 Exon 18 GAAACCCCTTATAGAAAACC 59 7797 7816 420 588521 2563 2582 Exon 18 GGAAACCCCTTATAGAAAAC 40 7798 7817 421 588522 2564 2583 Exon 18 AGGAAACCCCTTATAGAAAA 66 7799 7818 422 588523 2565 2584 Exon 18 CAGGAAACCCCTTATAGAAA 63 7800 7819 423 588524 2566 2585 Exon 18 GCAGGAAACCCCTTATAGAA 70 7801 7820 424 588525 2567 2586 Exon 18 AGCAGGAAACCCCTTATAGA 67 7802 7821 425 588526 2568 2587 Exon 18 CAGCAGGAAACCCCTTATAG 0 7803 7822 426 588527 2569 2588 Exon 18 CCAGCAGGAAACCCCTTATA 11 7804 7823 427 588528 2570 2589 Exon 18 TCCAGCAGGAAACCCCTTAT 15 7805 7824 428 532809 2571 2590 Exon 18 GTCCAGCAGGAAACCCCTTA 75 7806 7825 237 588529 2572 2591 Exon 18 TGTCCAGCAGGAAACCCCTT 16 7807 7826 429 588530 2573 2592 Exon 18 CTGTCCAGCAGGAAACCCCT 16 7808 7827 430 588531 2574 2593 Exon 18 CCTGTCCAGCAGGAAACCCC 19 7809 7828 431 588532 2575 2594 Exon 18 CCCTGTCCAGCAGGAAACCC 15 7810 7829 432 588533 2576 2595 Exon 18 CCCCTGTCCAGCAGGAAACC 29 7811 7830 433 532810 2577 2596 Exon 18 GCCCCTGTCCAGCAGGAAAC 74 7812 7831 238 588534 2578 2597 Exon 18 CGCCCCTGTCCAGCAGGAAA 21 7813 7832 434 588535 2579 2598 Exon 18 ACGCCCCTGTCCAGCAGGAA 16 7814 7833 435 588536 2580 2599 Exon 18 CACGCCCCTGTCCAGCAGGA 0 7815 7834 436 588537 2581 2600 Exon 18 CCACGCCCCTGTCCAGCAGG 8 7816 7835 437 588538 2582 2601 Exon 18 CCCACGCCCCTGTCCAGCAG 10 7817 7836 438 588539 2583 2602 Exon 18 TCCCACGCCCCTGTCCAGCA 23 7818 7837 439 588540 2584 2603 Exon 18 ATCCCACGCCCCTGTCCAGC 16 7819 7838 440 588541 2585 2604 Exon 18 AATCCCACGCCCCTGTCCAG 16 7820 7839 441 588542 2586 2605 Exon 18 CAATCCCACGCCCCTGTCCA 12 7821 7840 442 588543 2587 2606 Exon 18 TCAATCCCACGCCCCTGTCC 26 7822 7841 443 588544 2588 2607 Exon 18 TTCAATCCCACGCCCCTGTC 26 7823 7842 444 588545 2589 2608 Exon 18 ATTCAATCCCACGCCCCTGT 31 7824 7843 445 588546 2590 2609 Exon 18 AATTCAATCCCACGCCCCTG 22 7825 7844 446 588547 2591 2610 Exon 18 TAATTCAATCCCACGCCCCT 12 7826 7845 447 588548 2592 2611 Exon 18 TTAATTCAATCCCACGCCCC 20 7827 7846 448 588549 2593 2612 Exon 18 TTTAATTCAATCCCACGCCC 26 7828 7847 449 588550 2594 2613 Exon 18 TTTTAATTCAATCCCACGCC 32 7829 7848 450 588551 2595 2614 Exon 18 GTTTTAATTCAATCCCACGC 48 7830 7849 451 588552 2596 2615 Exon 18 TGTTTTAATTCAATCCCACG 57 7831 7850 452 588553 2597 2616 Exon 18 CTGTTTTAATTCAATCCCAC 49 7832 7851 453 588554 2598 2617 Exon 18 GCTGTTTTAATTCAATCCCA 64 7833 7852 454 532811 2599 2618 Exon 18 AGCTGTTTTAATTCAATCCC 78 7834 7853 239 588555 2600 2619 Exon 18 CAGCTGTTTTAATTCAATCC 48 7835 7854 455 588556 2601 2620 Exon 18 GCAGCTGTTTTAATTCAATC 55 7836 7855 456 588557 2602 2621 Exon 18 CGCAGCTGTTTTAATTCAAT 51 7837 7856 457 588558 2603 2622 Exon 18 TCGCAGCTGTTTTAATTCAA 51 7838 7857 458 532917 2604 2623 Exon 18 GTCGCAGCTGTTTTAATTCA 82 7839 7858 317 588559 2605 2624 Exon 18 TGTCGCAGCTGTTTTAATTC 58 7840 7859 459 588560 2606 2625 Exon 18 TTGTCGCAGCTGTTTTAATT 72 7841 7860 460 588561 2607 2626 Exon 18 GTTGTCGCAGCTGTTTTAAT 75 7842 7861 461 532952 2608 2627 Exon 18 TGTTGTCGCAGCTGTTTTAA 39 7843 7862 395 588562 2609 2628 Exon 18/ TTGTTGTCGCAGCTGTTTTA 53 n/a n/a 462 Repeat 588563 2610 2629 Exon 18/ TTTGTTGTCGCAGCTGTTTT 62 n/a n/a 463 Repeat 588564 2611 2630 Exon 18/ TTTTGTTGTCGCAGCTGTTT 63 n/a n/a 464 Repeat 588565 2612 2631 Exon 18/ TTTTTGTTGTCGCAGCTGTT 64 n/a n/a 465 Repeat

Example 122: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells by 5-10-5 MOE gapmers

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.313 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM, or 10.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. CFB mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 148 0.313 0.625 1.25 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532614 7 13 43 72 65 71 2.2 532635 12 0 3 28 0 0 >10 532692 26 0 12 52 55 74 3.7 532770 21 18 32 73 64 88 1.8 532775 8 0 26 35 47 59 6.2 532800 0 5 30 65 50 75 3.1 532809 12 30 28 40 46 66 4.6 532810 28 44 32 69 84 95 1.2 532811 66 83 90 94 97 99 <0.3 532917 64 85 88 96 97 99 <0.3 532952 50 53 68 80 91 94 0.4

Example 123: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a number of experiments with similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. CFB mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 149 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532811 19 53 81 87 96 97 0.2 588834 7 42 64 92 98 98 0.5 588835 11 30 66 89 97 97 0.5 588836 14 40 61 91 97 97 0.5 588837 6 39 67 89 96 97 0.5 588838 0 27 41 81 87 97 1.0 588842 17 51 68 86 93 95 0.3 588843 21 38 72 90 95 96 0.4 588870 9 31 56 88 95 97 0.6 588871 14 25 47 79 93 97 0.7 588872 18 28 59 84 92 97 0.6

TABLE 150 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532811 31 70 89 94 97 97 0.1 588844 31 60 77 91 95 96 0.1 588846 32 52 78 89 95 97 0.2 588847 22 52 77 91 95 97 0.2 588848 20 40 73 91 96 98 0.3 588851 40 52 82 94 97 97 0.1 588854 17 55 59 84 94 96 0.4 588855 10 32 56 82 93 96 0.6 588856 13 46 75 90 96 97 0.3 588857 11 52 73 94 96 97 0.3 588858 19 48 75 94 97 98 0.3

TABLE 151 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532811 42 66 88 96 97 98 0.1 588859 18 46 66 90 96 97 0.4 588860 55 80 94 97 97 97 <0.1 588861 24 61 86 93 96 97 0.2 588862 25 64 85 94 96 98 0.1 588863 50 73 89 96 96 98 <0.1 588864 52 80 92 96 98 98 <0.1 588865 46 72 91 96 96 99 <0.1 588866 47 76 88 96 97 98 <0.1 588867 43 69 83 92 96 99 0.1 588868 43 56 65 84 93 97 0.1

TABLE 152 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532810 0 14 38 72 89 96 1.2 532811 18 54 79 93 96 97 0.3 532952 19 34 73 87 94 96 0.4 588534 17 13 44 77 93 97 0.9 588544 12 43 69 86 89 93 0.4 588545 17 55 67 86 91 93 0.3 588546 10 32 67 85 91 93 0.6 588552 27 54 76 90 94 97 0.2 588553 32 68 87 93 95 97 0.1 588560 16 54 76 90 94 96 0.3 588561 18 45 68 85 93 96 0.4

TABLE 153 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532811 22 60 82 94 97 98 0.2 588536 2 38 65 90 96 97 0.6 588537 12 38 63 87 94 97 0.5 588547 19 35 61 86 93 97 0.5 588548 19 36 75 88 95 96 0.4 588554 0 76 92 95 97 97 <0.1 588555 31 61 89 96 97 98 0.1 588556 33 56 82 95 94 97 0.1 588562 12 39 71 87 94 97 0.4 588563 25 48 72 86 94 96 0.3 588564 15 33 63 89 91 97 0.5

TABLE 154 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 532811 39 68 86 96 98 98 0.1 588538 0 40 82 94 97 98 0.3 588539 34 65 88 95 98 98 0.1 588540 30 51 81 91 97 98 0.2 588549 31 57 82 95 96 98 0.1 588550 34 65 88 96 98 98 0.1 588551 47 66 87 96 98 99 <0.1 588557 40 84 95 98 98 98 <0.1 588558 45 73 93 97 98 99 <0.1 588559 51 69 83 96 98 99 <0.1 588565 19 56 81 92 96 98 0.2

Example 124: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a number of experiments with similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.06 μM, 0.25 μM, 1.00 μM, and 4.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. CFB mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 155 IC₅₀ ISIS No 0.06 μM 0.25 μM 1.00 μM 4.00 μM (μM) 532917 31 58 87 92 0.2 588860 18 50 79 93 0.3 599001 16 28 69 90 0.5 599024 14 32 74 90 0.4 599025 0 31 56 92 0.7 599032 28 44 62 88 0.3 599033 28 46 80 92 0.2 599077 8 20 59 80 0.8 599080 9 33 48 76 0.9 599086 7 22 53 83 0.8 599087 21 31 74 87 0.4 599088 13 37 69 82 0.5 599089 3 36 55 79 0.7 599093 25 59 79 88 0.2 599094 19 29 75 89 0.4 599095 29 43 67 87 0.3 599096 23 51 70 88 0.3 599149 20 53 82 92 0.3 599188 0 21 62 85 0.8

TABLE 156 IC₅₀ ISIS No 0.06 μM 0.25 μM 1.00 μM 4.00 μM (μM) 532917 0 42 81 91 0.4 588860 17 49 74 92 0.3 599155 29 52 67 87 0.3 599198 3 25 64 89 0.6 599201 13 26 67 91 0.5 599202 0 44 72 87 0.5 599203 22 41 75 88 0.3 599314 12 34 71 84 0.5 599316 7 37 66 88 0.5 599317 8 1 54 83 1.0 599321 8 33 70 85 0.5 599322 24 38 66 87 0.4 599327 22 32 66 89 0.4 599328 0 31 59 88 0.7 599330 5 43 67 84 0.5 599374 23 42 80 91 0.3 599378 21 57 80 93 0.2 599380 23 56 82 93 0.2 599432 17 37 73 93 0.4

TABLE 157 IC₅₀ ISIS No 0.06 μM 0.25 μM 1.00 μM 4.00 μM (μM) 532917 23 65 76 93 0.2 588860 17 60 76 90 0.3 601282 48 68 81 88 0.1 601269 18 59 80 94 0.2 601276 34 64 81 91 0.1 601275 14 39 78 90 0.4 601344 52 84 92 94 <0.06 601383 53 81 86 94 <0.06 601382 41 76 88 94 0.1 601385 52 74 89 91 <0.06 601332 41 69 86 94 0.1 601345 36 75 86 95 0.1 601371 34 72 91 93 0.1 601384 50 78 91 95 <0.06 601380 28 57 83 92 0.2 601387 48 61 82 88 0.1 601341 28 65 83 91 0.2 601346 31 69 82 93 0.1 601335 24 56 85 92 0.2

TABLE 158 IC₅₀ ISIS No 0.06 μM 0.25 μM 1.00 μM 4.00 μM (μM) 532917 31 66 86 93 0.1 588860 28 62 85 94 0.2 599208 24 50 71 89 0.3 599261 31 49 81 94 0.2 599267 41 48 80 88 0.2 599268 28 56 75 92 0.2 599313 14 24 71 92 0.5 599441 24 57 80 87 0.2 599494 13 55 86 94 0.3 599552 30 69 93 95 0.1 599553 34 71 93 96 0.1 599554 30 74 93 96 0.1 599568 40 77 90 97 0.1 599570 61 82 93 96 <0.06 599577 18 62 81 93 0.2 599581 27 60 80 94 0.2 599591 49 74 93 96 <0.06 599592 46 76 90 94 0.1 599593 44 72 91 95 0.1

TABLE 159 IC₅₀ ISIS No 0.06 μM 0.25 μM 1.00 μM 4.00 μM (μM) 532917 25 56 84 92 0.2 588860 11 51 80 92 0.3 599547 23 60 82 90 0.2 599569 42 73 85 88 0.1 599578 29 49 82 89 0.2 599582 21 56 78 91 0.2 599590 24 62 80 90 0.2 601209 21 49 85 88 0.3 601210 34 64 86 92 0.1 601212 46 68 88 90 0.1 601213 54 80 90 92 <0.06 601214 38 77 88 95 0.1 601215 42 64 85 92 0.1 601216 45 57 76 89 0.1 601264 29 58 86 95 0.2 601278 51 82 83 93 <0.06 601279 44 80 92 96 0.1 601280 44 73 87 94 0.1 601281 51 80 91 94 <0.06

Example 125: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. Additionally, a deoxy, MOE and (S)-cEt oligonucleotide, ISIS 594430, was designed with the same sequence (CTCCTTCCGAGTCAGC, SEQ ID NO: 549) and target region (target start site 2195 of SEQ ID NO: 1 and target start site 6983 of SEQ ID NO: 2) as ISIS 588870, another deoxy, MOE, and (S)-cEt oligonucleotide. ISIS 594430 is a 3-10-3 (S)-cEt gapmer.

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.01 μM, 0.04 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. CFB mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 160 0.01 0.04 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No μM μM μM μM μM μM μM (μM) 588536 0 0 0 5 45 73 94 1.4 588548 0 0 0 19 52 78 90 1.2 588553 0 0 9 42 76 85 94 0.6 588555 0 52 23 58 78 83 95 0.3 588847 4 1 18 45 67 84 96 0.5 588848 0 3 13 38 67 83 95 0.6 594430 0 0 10 34 50 55 84 1.4

Example 126: Tolerability of MOE Gapmers Targeting Human CFB in CD1 Mice

CD1® mice (Charles River, MA) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Study 1 (with 5-10-5 MOE Gapmers)

Groups of seven-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotide. A group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. One group of mice was injected with subcutaneously once a week for 6 weeks with 100 mg/kg of control oligonucleotide ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, designated herein as SEQ ID NO: 809, 5-10-5 MOE gapmer with no known murine target). Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 161 Plasma chemistry markers in CD1 mice plasma on day 40 ALT AST BUN (IU/L) (IU/L) (mg/dL) PBS 25 46 20 ISIS 532614 513 407 22 ISIS 532692 131 130 24 ISIS 532770 36 53 25 ISIS 532775 193 158 23 ISIS 532800 127 110 25 ISIS 532809 36 42 22 ISIS 532810 229 286 26 ISIS 532811 197 183 21 ISIS 532917 207 204 27 ISIS 532952 246 207 25 ISIS 141923 39 67 23 Weights

Body weights of the mice were measured on day 40 before sacrificing the mice. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 162 Weights (g) of CD1 mice on day 40 Body Kidney Liver Spleen PBS 44 0.8 2.0 0.1 ISIS 532614 43 0.7 4.3 0.2 ISIS 532692 42 0.7 2.6 0.2 ISIS 532770 42 0.6 2.3 0.2 ISIS 532775 42 0.7 2.5 0.2 ISIS 532800 43 0.6 2.8 0.3 ISIS 532809 42 0.6 2.2 0.1 ISIS 532810 43 0.6 2.3 0.2 ISIS 532811 41 0.7 2.4 0.2 ISIS 532917 42 0.7 3.0 0.2 ISIS 532952 44 0.8 2.5 0.3 ISIS 141923 41 0.6 2.0 0.1 Study 2 (with 5-10-5 MOE Gapmers)

Groups of six- to eight-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotide. Two groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with PBS. One group of mice was injected with subcutaneously once a week for 6 weeks with 100 mg/kg of control oligonucleotide ISIS 141923. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 163 Plasma chemistry markers in CD1 mice plasma on day 45 ALT AST Albumin BUN (IU/L) (IU/L) (g/dL) (mg/dL) PBS 39 53 2.9 29 PBS 50 97 2.9 30 ISIS 141923 163 174 4.1 25 ISIS 532810 321 297 2.5 26 ISIS 532952 182 199 2.7 27 ISIS 588534 276 248 2.6 29 ISIS 588536 48 60 2.9 31 ISIS 588537 72 79 4.0 25 ISIS 588538 63 67 4.5 29 ISIS 588539 238 177 3.9 28 ISIS 588545 496 256 4.4 24 ISIS 588547 323 210 4.4 25 ISIS 588548 61 63 4.2 27 ISIS 588549 127 132 4.1 23 ISIS 588551 302 282 4.2 22 ISIS 588552 76 98 4.0 30 ISIS 588558 1066 521 3.9 27 ISIS 588559 76 94 4.1 26 ISIS 588561 502 500 4.4 26 ISIS 588563 50 99 4.4 28 Weights

Body weights of the mice were measured on day 42. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 45. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 164 Weights (g) of CD1 mice on day 40 Body Kidney Liver Spleen PBS 44 0.7 2.4 0.1 PBS 43 0.7 2.4 0.2 ISIS 141923 43 0.6 2.4 0.2 ISIS 532810 41 0.6 1.9 0.1 ISIS 532952 43 0.6 2.4 0.2 ISIS 588534 44 0.7 2.8 0.2 ISIS 588536 43 0.7 2.7 0.2 ISIS 588537 43 0.7 2.4 0.2 ISIS 588538 44 0.7 2.8 0.2 ISIS 588539 44 0.6 2.7 0.2 ISIS 588545 44 0.8 3.3 0.3 ISIS 588547 42 0.6 3.3 0.3 ISIS 588548 43 0.6 2.8 0.2 ISIS 588549 42 0.6 2.8 0.3 ISIS 588551 39 0.6 2.2 0.2 ISIS 588552 41 0.6 2.2 0.2 ISIS 588558 44 0.7 3.3 0.3 ISIS 588559 43 0.6 2.7 0.3 ISIS 588561 40 0.7 2.4 0.3 ISIS 588563 41 0.7 2.4 0.2 Study 3 (with 5-10-5 MOE Gapmers)

Groups of six- to eight-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotide. Two groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 165 Plasma chemistry markers in CD1 mice plasma on day 42 ALT AST Albumin BUN (IU/L) (IU/L) (g/dL) (mg/dL) PBS 37 108 3.1 30 PBS 45 51 3.0 27 ISIS 588544 209 168 2.9 26 ISIS 588546 526 279 3.0 22 ISIS 588550 82 136 2.7 25 ISIS 588553 79 105 3.0 24 ISIS 588554 112 220 3.2 19 ISIS 588555 95 162 2.8 25 ISIS 588556 345 236 3.0 26 ISIS 588557 393 420 2.8 24 ISIS 588560 109 148 2.7 27 ISIS 588562 279 284 2.8 22 ISIS 588564 152 188 3.0 23 ISIS 588565 247 271 2.8 28 Weights

Body weights of the mice were measured on day 42. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 42. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 166 Weights (g) of CD1 mice on day 40 Body Kidney Liver Spleen PBS 42 0.7 2.4 0.1 PBS 41 0.7 2.4 0.2 ISIS 588544 44 0.6 1.9 0.1 ISIS 588546 43 0.6 2.4 0.2 ISIS 588550 41 0.7 2.8 0.2 ISIS 588553 44 0.7 2.7 0.2 ISIS 588554 40 0.7 2.4 0.2 ISIS 588555 44 0.7 2.8 0.2 ISIS 588556 39 0.6 2.7 0.2 ISIS 588557 41 0.8 3.3 0.3 ISIS 588560 38 0.6 3.2 0.3 ISIS 588562 41 0.6 2.8 0.2 ISIS 588564 40 0.6 2.8 0.3 ISIS 588565 39 0.6 2.2 0.2 Study 4 (with (S) cEt Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups often-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide from the studies described above. In addition, two oligonucleotides, ISIS 594431 and ISIS 594432, were designed as 3-10-3 (S)-cEt gapmers and were also tested in this study. ISIS 594431 (ACCTCCTTCCGAGTCA, SEQ ID NO: 550) targets the same region as ISIS 588871, a deoxy, MOE and (S)-cEt gapmer (target start site 2197 of SEQ ID NO: 1 and target start site 6985 of SEQ ID NO: 2). ISIS 594432 (TGGTCACATTCCCTTC, SEQ ID NO: 542) targets the same region as ISIS 588872 a deoxy, MOE and (S)-cEt gapmer (target start site 154 of SEQ ID NO: 1 and target start site 1875 of SEQ ID NO: 2).

Two groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 167 Plasma chemistry markers in CD1 mice plasma on day 42 ALT AST Albumin Creatinine BUN Chemistry (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) PBS — 71 77 2.7 0.2 29 PBS — 30 36 2.7 0.2 26 ISIS 588834 Deoxy, MOE and (S)-cEt 436 510 2.8 0.2 25 ISIS 588835 Deoxy, MOE and (S)-cEt 70 98 3.0 0.2 27 ISIS 588836 Deoxy, MOE and (S)-cEt 442 312 2.7 0.2 27 ISIS 588846 Deoxy, MOE and (S)-cEt 50 75 2.5 0.1 28 ISIS 588847 Deoxy, MOE and (S)-cEt 44 71 2.6 0.1 24 ISIS 588848 Deoxy, MOE and (S)-cEt 47 70 2.4 0.1 27 ISIS 588857 Deoxy, MOE and (S)-cEt 1287 655 2.7 0.2 26 ISIS 588858 Deoxy, MOE and (S)-cEt 1169 676 2.5 0.2 26 ISIS 588859 Deoxy, MOE and (S)-cEt 1036 1300 3.2 0.2 25 ISIS 588861 Deoxy, MOE and (S)-cEt 749 466 3.1 0.1 24 ISIS 588862 Deoxy, MOE and (S)-cEt 1564 1283 2.9 0.2 22 ISIS 588863 Deoxy, MOE and (S)-cEt 477 362 2.8 0.1 23 ISIS 588864 Deoxy, MOE and (S)-cEt 118 165 2.9 0.2 27 ISIS 588866 Deoxy, MOE and (S)-cEt 843 784 3.2 0.2 25 ISIS 594430 3-10-3 (S)-cEt 89 99 2.4 0.1 28 ISIS 594431 3-10-3 (S)-cEt 590 433 3.0 0.2 24 ISIS 594432 3-10-3 (S)-cEt 2595 2865 2.4 0.1 25 Weights

Body weights of the mice were measured on day 39. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 42. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 168 Weights (g) of CD1 mice Chemistry Body Kidney Liver Spleen PBS — 37 0.6 2.1 0.1 PBS — 45 0.7 2.5 0.2 ISIS 588834 Deoxy, MOE and (S)-cEt 40 0.6 3.2 0.2 ISIS 588835 Deoxy, MOE and (S)-cEt 38 0.7 2.8 0.3 ISIS 588836 Deoxy, MOE and (S)-cEt 41 0.7 2.3 0.2 ISIS 588837 Deoxy, MOE and (S)-cEt 38 0.6 2.4 0.3 ISIS 588846 Deoxy, MOE and (S)-cEt 39 0.6 2.3 0.2 ISIS 588847 Deoxy, MOE and (S)-cEt 40 0.7 2.5 0.2 ISIS 588848 Deoxy, MOE and (S)-cEt 43 0.7 2.6 0.3 ISIS 588857 Deoxy, MOE and (S)-cEt 39 0.6 3.3 0.2 ISIS 588858 Deoxy, MOE and (S)-cEt 37 0.6 3.4 0.2 ISIS 588859 Deoxy, MOE and (S)-cEt 41 0.7 2.5 0.3 ISIS 588861 Deoxy, MOE and (S)-cEt 39 0.6 2.6 0.4 ISIS 588862 Deoxy, MOE and (S)-cEt 34 0.6 2.5 0.4 ISIS 588863 Deoxy, MOE and (S)-cEt 40 0.6 2.7 0.3 ISIS 588864 Deoxy, MOE and (S)-cEt 40 0.7 2.3 0.2 ISIS 588866 Deoxy, MOE and (S)-cEt 45 0.7 3.0 0.2 ISIS 594430 3-10-3 (S)-cEt 39 0.6 2.2 0.2 ISIS 594431 3-10-3 (S)-cEt 36 0.6 3.2 0.2 ISIS 594432 3-10-3 (S)-cEt 31 0.4 1.9 0.1 Study 5 (with MOE Gapmers, (S) cEt Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide. Two groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 169 Plasma chemistry markers in CD1 mice plasma on day 42 ALT AST Albumin Creatinine BUN Chemistry (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) PBS — 33 84 2.9 0.2 28 PBS — 32 65 2.5 0.1 27 ISIS 532692 5-10-5 MOE 363 281 3.0 0.2 30 ISIS 532770 5-10-5 MOE 69 100 2.9 0.1 28 ISIS 532775 5-10-5 MOE 371 333 2.6 0.1 29 ISIS 532800 5-10-5 MOE 104 106 2.7 0.1 31 ISIS 532809 5-10-5 MOE 69 127 2.8 0.1 26 ISIS 588540 5-10-5 MOE 66 110 2.8 0.1 26 ISIS 588838 3-10-3 (S)-cEt 391 330 2.9 0.1 25 ISIS 588842 Deoxy, MOE and (S)-cEt 224 264 2.6 0.1 26 ISIS 588843 3-10-3 (S)-cEt 185 160 2.8 0.1 24 ISIS 588844 Deoxy, MOE and (S)-cEt 304 204 2.7 0.1 25 ISIS 588851 Deoxy, MOE and (S)-cEt 186 123 2.7 0.1 31 ISIS 588854 Deoxy, MOE and (S)-cEt 1232 925 2.7 0.1 25 ISIS 588855 Deoxy, MOE and (S)-cEt 425 321 2.7 0.1 28 ISIS 588856 Deoxy, MOE and (S)-cEt 78 101 2.4 0.1 31 ISIS 588865 Deoxy, MOE and (S)-cEt 126 145 2.5 0.1 23 ISIS 588867 Deoxy, MOE and (S)-cEt 108 112 2.5 0.1 32 ISIS 588868 Deoxy, MOE and (S)-cEt 61 124 2.5 0.1 28 ISIS 588870 Deoxy, MOE and (S)-cEt 48 69 2.4 0.1 31 ISIS 588871 Deoxy, MOE and (S)-cEt 723 881 2.5 0.1 24 ISIS 588872 Deoxy, MOE and (S)-cEt 649 654 2.7 0.1 26 Weights

Body weights of the mice were measured on day 40. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 42. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 170 Weights (g) of CD1 mice Chemistry Body Kidney Liver Spleen PBS — 46 0.7 2.3 0.2 PBS — 44 0.7 2.3 0.2 ISIS 532692 5-10-5 MOE 44 0.6 2.8 0.2 ISIS 532770 5-10-5 MOE 43 0.6 2.2 0.2 ISIS 532775 5-10-5 MOE 43 0.6 2.8 0.2 ISIS 532800 5-10-5 MOE 47 0.7 2.9 0.2 ISIS 532809 5-10-5 MOE 44 0.7 2.6 0.2 ISIS 588540 5-10-5 MOE 44 0.7 2.5 0.2 ISIS 588838 3-10-3 (S)-cEt 45 0.7 3.1 0.2 ISIS 588842 Deoxy, MOE and (S)-cEt 41 0.6 2.6 0.2 ISIS 588843 3-10-3 (S)-cEt 43 0.7 2.9 0.2 ISIS 588844 Deoxy, MOE and (S)-cEt 43 0.7 2.8 0.2 ISIS 588851 Deoxy, MOE and (S)-cEt 46 0.6 2.6 0.2 ISIS 588854 Deoxy, MOE and (S)-cEt 45 0.7 4.1 0.2 ISIS 588855 Deoxy, MOE and (S)-cEt 44 0.7 2.9 0.3 ISIS 588856 Deoxy, MOE and (S)-cEt 44 0.7 3.2 0.2 ISIS 588865 Deoxy, MOE and (S)-cEt 45 0.7 2.6 0.3 ISIS 588867 Deoxy, MOE and (S)-cEt 46 0.7 3.2 0.3 ISIS 588868 Deoxy, MOE and (S)-cEt 42 0.7 2.9 0.3 ISIS 588870 Deoxy, MOE and (S)-cEt 43 0.6 2.2 0.2 ISIS 588871 Deoxy, MOE and (S)-cEt 41 0.7 3.1 0.2 ISIS 588872 Deoxy, MOE and (S)-cEt 39 0.6 3.2 0.3 Study 6 (with Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 50 mg/kg of deoxy, MOE, and (S)-cEt oligonucleotides. Two groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, creatinine, bilirubin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 171 Plasma chemistry markers in CD1 mice plasma on day 45 ALT AST Albumin Creatinine Bilirubin BUN (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 39 78 3.4 0.2 0.2 31 PBS 37 59 2.9 0.1 0.2 27 ISIS 599552 167 208 3.0 0.1 0.2 32 ISIS 599553 43 86 2.9 0.1 0.2 28 ISIS 599554 57 101 2.2 0.2 0.2 31 ISIS 599569 469 530 3.5 0.2 0.3 27 ISIS 599577 37 84 2.9 0.1 0.1 31 ISIS 599578 45 104 2.8 0.1 0.2 30 ISIS 599581 54 88 3.1 0.1 0.2 31 ISIS 599590 1741 1466 3.1 0.1 0.3 25 ISIS 599591 2230 1183 3.2 0.1 0.3 27 ISIS 601209 68 104 2.9 0.1 0.2 30 ISIS 601212 1795 968 3.2 0.1 0.3 22 ISIS 601215 424 385 3.1 0.1 0.4 25 ISIS 601216 90 125 2.9 0.1 0.2 29 ISIS 601276 946 366 2.9 0.1 0.5 31 ISIS 601282 831 540 3.3 0.2 0.2 32 Weights

Body weights of the mice were measured on day 40. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 45. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 172 Weights (g) of CD1 mice Body Kidney Liver Spleen PBS 40 0.7 2.1 0.2 PBS 42 0.8 2.3 0.2 ISIS 599552 38 0.6 2.3 0.2 ISIS 599553 39 0.7 2.2 0.2 ISIS 599554 39 0.7 2.4 0.2 ISIS 599569 39 0.7 2.2 0.2 ISIS 599577 41 0.7 2.5 0.2 ISIS 599578 37 0.6 2.0 0.2 ISIS 599581 40 0.6 2.5 0.2 ISIS 599590 34 0.6 3.5 0.2 ISIS 599591 38 0.8 2.7 0.2 ISIS 601209 42 0.7 2.6 0.3 ISIS 601212 38 0.6 2.9 0.2 ISIS 601215 36 0.7 2.6 0.2 ISIS 601216 42 0.6 2.7 0.2 ISIS 601276 42 0.7 3.2 0.2 ISIS 601282 38 0.7 3.2 0.2 Study 7 (with MOE Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotides. One group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 173 Plasma chemistry markers in CD1 mice plasma on day 45 ALT AST Albumin Creatinine BUN Chemistry (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) PBS — 120 102 2.7 0.2 26 ISIS 588842 Deoxy, MOE and (S)-cEt 177 164 2.7 0.1 23 ISIS 588843 Deoxy, MOE and (S)-cEt 98 194 2.7 0.1 24 ISIS 588851 Deoxy, MOE and (S)-cEt 91 142 2.6 0.1 23 ISIS 588856 Deoxy, MOE and (S)-cEt 78 110 2.7 0.1 23 ISIS 599024 3-10-4 MOE 91 108 2.7 0.1 23 ISIS 599087 5-7-5 MOE 198 183 2.6 0.2 28 ISIS 599093 5-7-5 MOE 3285 2518 2.6 0.2 24 ISIS 599149 4-8-5 MOE 30 64 2.9 0.2 25 ISIS 599155 4-8-5 MOE 145 189 2.6 0.2 25 ISIS 599202 5-8-5 MOE 150 128 2.8 0.2 23 ISIS 599203 5-8-5 MOE 111 127 2.8 0.2 24 ISIS 599208 5-8-5 MOE 146 178 2.9 0.2 22 ISIS 599261 3-10-5 MOE 144 165 2.8 0.2 26 ISIS 599267 3-10-5 MOE 96 132 2.6 0.2 27 ISIS 599268 3-10-5 MOE 87 115 2.6 0.1 23 ISIS 599322 6-7-6 MOE 115 138 2.7 0.1 22 ISIS 599374 5-9-5 MOE 375 271 2.6 0.1 21 ISIS 599378 5-9-5 MOE 77 99 2.7 0.1 23 ISIS 599441 6-8-6 MOE 150 250 2.9 0.1 23 Weights

Body weights of the mice were measured on day 44. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 49. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 174 Weights (g) of CD1 mice Chemistry Body Kidney Liver Spleen PBS — 39 0.6 1.9 0.1 ISIS 588842 Deoxy, MOE and (S)-cEt 38 0.5 2.1 0.1 ISIS 588843 Deoxy, MOE and (S)-cEt 41 0.6 2.4 0.2 ISIS 588851 Deoxy, MOE and (S)-cEt 42 0.6 2.2 0.2 ISIS 588856 Deoxy, MOE and (S)-cEt 42 0.7 2.6 0.2 ISIS 599024 3-10-4 MOE 41 0.6 4.0 0.2 ISIS 599087 5-7-5 MOE 44 0.8 2.6 0.3 ISIS 599093 5-7-5 MOE 39 0.6 2.3 0.2 ISIS 599149 4-8-5 MOE 42 0.7 2.8 0.2 ISIS 599155 4-8-5 MOE 41 0.7 2.1 0.2 ISIS 599202 5-8-5 MOE 43 0.6 2.6 0.2 ISIS 599203 5-8-5 MOE 42 0.6 2.6 0.2 ISIS 599208 5-8-5 MOE 40 0.6 2.1 0.2 ISIS 599261 3-10-5 MOE 39 0.7 3.4 0.3 ISIS 599267 3-10-5 MOE 42 0.8 2.5 0.3 ISIS 599268 3-10-5 MOE 41 0.7 2.1 0.2 ISIS 599322 6-7-6 MOE 43 0.6 2.2 0.2 ISIS 599374 5-9-5 MOE 37 0.6 2.2 0.2 ISIS 599378 5-9-5 MOE 43 0.7 2.7 0.2 ISIS 599441 6-8-6 MOE 42 0.6 2.5 0.3 Study 8 (with MOE Gapmers, Deoxy, MOE and (S)-cEt Oligonucleotides, and (S)-cEt Gapmers)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers, or 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotides or (S)-cEt gapmers. One group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, albumin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below.

TABLE 175 Plasma chemistry markers in CD1 mice plasma on day 43 Dose ALT AST Albumin Creatinine BUN Chemistry (mg/kg/wk) (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) PBS — — 37 57 2.5 0.08 26 ISIS 532770 5-10-5 MOE 100 57 73 2.5 0.07 24 ISIS 532800 5-10-5 MOE 100 74 126 2.8 0.10 26 ISIS 532809 5-10-5 MOE 100 83 73 2.5 0.07 23 ISIS 588540 5-10-5 MOE 100 106 102 2.7 0.09 27 ISIS 588544 5-10-5 MOE 100 66 62 2.6 0.10 24 ISIS 588548 5-10-5 MOE 100 48 67 2.6 0.08 23 ISIS 588550 5-10-5 MOE 100 65 106 2.5 0.10 25 ISIS 588553 5-10-5 MOE 100 78 90 2.6 0.09 25 ISIS 588555 5-10-5 MOE 100 94 89 2.5 0.08 23 ISIS 588848 Deoxy, MOE 50 38 54 2.3 0.07 25 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 50 63 72 2.5 0.10 27 Weights

Body weights of the mice were measured on day 36. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 43. The results for the organ weights were expressed as a ratio to the body weights and normalized to the PBS control ratio.

TABLE 176 Organ Weights/Body weight (BW) of CD1 mice Dose Kidney/ Liver/ Spleen/ Chemistry (mg/kg/wk) BW BW BW PBS — — 1.0 1.0 1.0 ISIS 532770 5-10-5 MOE 100 1.4 1.1 1.0 ISIS 532800 5-10-5 MOE 100 1.5 1.1 0.9 ISIS 532809 5-10-5 MOE 100 1.3 1.2 0.9 ISIS 588540 5-10-5 MOE 100 1.3 1.2 1.0 ISIS 588544 5-10-5 MOE 100 1.6 1.1 1.0 ISIS 588548 5-10-5 MOE 100 1.7 1.2 1.0 ISIS 588550 5-10-5 MOE 100 1.5 1.2 1.0 ISIS 588553 5-10-5 MOE 100 1.5 1.0 0.8 ISIS 588555 5-10-5 MOE 100 1.8 1.2 1.0 ISIS 588848 Deoxy, MOE 50 1.3 1.0 0.9 and (S)-cEt ISIS 594430 3-10-3 (S)- 50 1.4 1.1 0.9 cEt Cytokine Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for measurements of the various cytokine levels, such as IL-6, MDC, MIP1β, IP-10, MCP1, MIP-1α, and RANTES. The results are presented in Table 54.

TABLE 177 Cytokine levels (pg/mL) in CD1 mice plasma Chemistry IL-6 MDC MIP1β IP-10 MCP1 MlP-1α RANTES PBS — 70 16 23 20 17 6 2 ISIS 532770 5-10-5 MOE 101 18 146 116 101 24 6 ISIS 532800 5-10-5 MOE 78 17 83 53 105 1 3 ISIS 532809 5-10-5 MOE 66 19 60 32 55 20 4 ISIS 588540 5-10-5 MOE 51 18 126 70 75 4 3 ISIS 588544 5-10-5 MOE 157 14 94 34 102 1 3 ISIS 588548 5-10-5 MOE 164 12 90 66 84 10 4 ISIS 588550 5-10-5 MOE 58 21 222 124 157 3 5 ISIS 588553 5-10-5 MOE 62 14 183 60 103 9 4 ISIS 588555 5-10-5 MOE 70 19 172 171 178 16 9 ISIS 588848 Deoxy, MOE 59 13 61 27 63 12 4 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 48 14 56 38 85 10 3 Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for measurements of hematocrit (HCT), as well as of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin (Hb) content. The results are presented in Table 55.

TABLE 178 Hematology markers in CD1 mice plasma HCT Hb WBC RBC Platelets Chemistry (%) (g/dL) (10³/μL) (10⁶/μL) (10³/μL) PBS — 46 15 7 9 960 ISIS 532770 5-10-5 MOE 45 14 5 9 879 ISIS 532800 5-10-5 MOE 45 14 5 9 690 ISIS 532809 5-10-5 MOE 46 14 6 9 1005 ISIS 588540 5-10-5 MOE 49 15 6 10 790 ISIS 588544 5-10-5 MOE 36 11 7 7 899 ISIS 588548 5-10-5 MOE 46 14 6 9 883 ISIS 588550 5-10-5 MOE 42 13 8 8 721 ISIS 588553 5-10-5 MOE 45 14 6 9 719 ISIS 588555 5-10-5 MOE 43 13 8 9 838 ISIS 588848 Deoxy, MOE 40 15 8 10 840 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 45 14 8 9 993

Example 127: Tolerability of Antisense Oligonucleotides Targeting Human CFB in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Study 1 (with 5-10-5 MOE Gapmers)

Male Sprague-Dawley rats, seven- to eight-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of 5-10-5 MOE gapmers. One control group of 6 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 179 Liver function markers in Sprague-Dawley rats ALT AST (IU/L) (IU/L) PBS 66 134 ISIS 588544 101 329 ISIS 588550 69 157 ISIS 588553 88 304 ISIS 588554 202 243 ISIS 588555 94 113 ISIS 588556 102 117 ISIS 588560 206 317 ISIS 588564 292 594 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 180 Kidney function markers (mg/dL) in Sprague-Dawley rats BUN Creatinine PBS 18 3.5 ISIS 588544 21 3.1 ISIS 588550 21 3.0 ISIS 588553 22 2.8 ISIS 588554 23 3.0 ISIS 588555 22 3.5 ISIS 588556 21 3.2 ISIS 588560 26 2.4 ISIS 588564 24 2.7 Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 181 Weights (g) Body Liver Spleen Kidney PBS 422 16 1.2 3.9 ISIS 588544 353 15 1.7 2.9 ISIS 588550 321 14 2.1 3.2 ISIS 588553 313 15 2.3 3.2 ISIS 588554 265 11 1.6 2.7 ISIS 588555 345 14 1.4 3.3 ISIS 588556 328 13 1.7 3.1 ISIS 588560 270 13 2.4 3.0 ISIS 588564 253 12 2.9 3.0 Study 2 (with Deoxy, MOE and (S)-cEt Oligonucleotides)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of deoxy, MOE, and (S)-cEt oligonucleotides. Two control groups of 3 rats each were injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase), and albumin were measured and the results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 182 Liver function markers in Sprague-Dawley rats ALT AST Albumin (IU/L) (IU/L) (g/dL) PBS 55 150 3.4 PBS 64 91 3.5 ISIS 588554 52 92 3.2 ISIS 588835 971 844 4.1 ISIS 588842 317 359 3.8 ISIS 588843 327 753 2.9 ISIS 588846 70 111 3.2 ISIS 588847 65 100 3.0 ISIS 588864 91 109 3.0 ISIS 594430 85 106 3.7 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 183 Kidney function markers (mg/dL) in Sprague-Dawley rats BUN Creatinine PBS 17 0.4 PBS 21 0.4 ISIS 588554 20 0.4 ISIS 588835 23 0.5 ISIS 588842 22 0.4 ISIS 588843 51 0.4 ISIS 588846 25 0.5 ISIS 588847 23 0.5 ISIS 588864 23 0.4 ISIS 594430 22 0.5 Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 184 Weights (g) Body Liver Spleen Kidney PBS 466 16 0.9 3.8 PBS 485 16 0.9 3.6 ISIS 588554 393 15 2.3 2.6 ISIS 588835 387 16 1.0 3.3 ISIS 588842 414 22 1.5 3.7 ISIS 588843 427 20 2.5 4.2 ISIS 588846 366 16 2.1 3.3 ISIS 588847 402 15 1.6 3.1 ISIS 588864 364 15 2.1 3.8 ISIS 594430 420 16 1.2 3.6 Study 3 (with MOE Gapmers)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers. One control group of 6 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 43 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 185 Liver function markers in Sprague-Dawley rats ALT AST Albumin Chemistry (IU/L) (IU/L) (g/dL) PBS — 52 110 3.7 ISIS 588563 5-10-5 MOE 175 291 2.9 ISIS 599024 3-10-4 MOE 139 173 1.4 ISIS 599093 5-7-5 MOE 116 238 2.6 ISIS 599149 4-8-5 MOE 232 190 3.4 ISIS 599155 4-8-5 MOE 108 215 2.5 ISIS 599202 5-8-5 MOE 65 86 3.5 ISIS 599203 5-8-5 MOE 71 97 3.1 ISIS 599208 5-8-5 MOE 257 467 1.9 ISIS 599261 3-10-5 MOE 387 475 1.5 ISIS 599267 3-10-5 MOE 201 337 2.7 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 186 Kidney function markers (mg/dL) in Sprague-Dawley rats Chemistry BUN Creatinine PBS — 16 0.3 ISIS 588563 5-10-5 MOE 26 0.4 ISIS 599024 3-10-4 MOE 135 1.2 ISIS 599093 5-7-5 MOE 29 0.4 ISIS 599149 4-8-5 MOE 23 0.4 ISIS 599155 4-8-5 MOE 29 0.4 ISIS 599202 5-8-5 MOE 19 0.4 ISIS 599203 5-8-5 MOE 22 0.4 ISIS 599208 5-8-5 MOE 26 0.3 ISIS 599261 3-10-5 MOE 228 1.6 ISIS 599267 3-10-5 MOE 24 0.4 Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 187 Weights (g) Chemistry Body Liver Spleen Kidney PBS — 471 16 1.0 4.1 ISIS 588563 5-10-5 MOE 311 16 3.4 4.1 ISIS 599024 3-10-4 MOE 297 11 1.0 3.5 ISIS 599093 5-7-5 MOE 332 18 4.1 5.0 ISIS 599149 4-8-5 MOE 388 16 2.3 3.7 ISIS 599155 4-8-5 MOE 290 15 2.9 4.5 ISIS 599202 5-8-5 MOE 359 13 1.3 3.2 ISIS 599203 5-8-5 MOE 334 14 1.8 3.3 ISIS 599208 5-8-5 MOE 353 29 4.7 4.6 ISIS 599261 3-10-5 MOE 277 10 0.9 3.2 ISIS 599267 3-10-5 MOE 344 21 3.9 4.7 Study 4 (with MOE Gapmers)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers. One control group of 6 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 188 Liver function markers in Sprague-Dawley rats ALT AST Albumin Chemistry (IU/L) (IU/L) (g/dL) PBS — 48 77 3.9 ISIS 532800 5-10-5 MOE 72 111 3.4 ISIS 532809 5-10-5 MOE 59 89 3.8 ISIS 588540 5-10-5 MOE 146 259 3.8 ISIS 599268 3-10-5 MOE 175 206 2.7 ISIS 599322 6-7-6 MOE 523 567 3.3 ISIS 599374 5-9-5 MOE 114 176 3.0 ISIS 599378 5-9-5 MOE 124 116 3.2 ISIS 599380 5-9-5 MOE 148 210 3.4 ISIS 599441 6-8-6 MOE 51 91 2.6 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 189 Kidney function markers (mg/dL) in Sprague-Dawley rats Chemistry BUN Creatinine PBS — 15 0.4 ISIS 532800 5-10-5 MOE 26 0.5 ISIS 532809 5-10-5 MOE 18 0.5 ISIS 588540 5-10-5 MOE 22 0.5 ISIS 599268 3-10-5 MOE 28 0.5 ISIS 599322 6-7-6 MOE 24 0.5 ISIS 599374 5-9-5 MOE 29 0.5 ISIS 599378 5-9-5 MOE 22 0.4 ISIS 599380 5-9-5 MOE 26 0.5 ISIS 599441 6-8-6 MOE 24 0.4 Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 190 Weights (g) Chemistry Body Liver Spleen Kidney PBS — 502 16 0.9 3.7 ISIS 532800 5-10-5 MOE 376 16 2.0 3.4 ISIS 532809 5-10-5 MOE 430 16 1.4 3.4 ISIS 588540 5-10-5 MOE 391 16 1.8 3.5 ISIS 599268 3-10-5 MOE 332 16 3.6 3.6 ISIS 599322 6-7-6 MOE 348 13 2.1 3.4 ISIS 599374 5-9-5 MOE 302 12 2.0 3.3 ISIS 599378 5-9-5 MOE 332 11 1.1 2.8 ISIS 599380 5-9-5 MOE 350 11 1.5 3.3 ISIS 599441 6-8-6 MOE 368 16 2.5 3.3 Study 5 (with MOE Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmer or with 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotides. One control group of 4 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 191 Liver function markers in Sprague-Dawley rats ALT AST Albumin Chemistry (IU/L) (IU/L) (g/dL) PBS — 49 74 3.3 ISIS 532770 5-10-5 MOE 95 132 3.3 ISIS 588851 Deoxy, MOE, and (S)-cEt 47 72 3.1 ISIS 588856 Deoxy, MOE, and (S)-cEt 56 75 3.0 ISIS 588865 Deoxy, MOE, and (S)-cEt 62 84 2.9 ISIS 588867 Deoxy, MOE, and (S)-cEt 73 214 2.9 ISIS 588868 Deoxy, MOE, and (S)-cEt 59 83 3.1 ISIS 588870 Deoxy, MOE, and (S)-cEt 144 144 3.4 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma and urine levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Tables below, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 192 Kidney function markers (mg/dL) in the plasma of Sprague-Dawley rats Chemistry BUN Creatinine PBS — 18 0.3 ISIS 532770 5-10-5 MOE 20 0.4 ISIS 588851 Deoxy, MOE, and (S)-cEt 20 0.4 ISIS 588856 Deoxy, MOE, and (S)-cEt 22 0.4 ISIS 588865 Deoxy, MOE, and (S)-cEt 24 0.5 ISIS 588867 Deoxy, MOE, and (S)-cEt 22 0.4 ISIS 588868 Deoxy, MOE, and (S)-cEt 19 0.4 ISIS 588870 Deoxy, MOE, and (S)-cEt 20 0.5

TABLE 193 Kidney function markers (mg/dL) in the urine of Sprague-Dawley rats Total Chemistry protein Creatinine PBS — 80 92 ISIS 532770 5-10-5 MOE 466 69 ISIS 588851 Deoxy, MOE, and (S)-cEt 273 64 ISIS 588856 Deoxy, MOE, and (S)-cEt 259 68 ISIS 588865 Deoxy, MOE, and (S)-cEt 277 67 ISIS 588867 Deoxy, MOE, and (S)-cEt 337 68 ISIS 588868 Deoxy, MOE, and (S)-cEt 326 75 ISIS 588870 Deoxy, MOE, and (S)-cEt 388 82 Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 194 Weights (g) Chemistry Body Liver Spleen Kidney PBS — 489 16 0.9 3.5 ISIS 532770 5-10-5 MOE 372 15 1.7 3.1 ISIS 588851 Deoxy, MOE, 285 14 1.4 3.2 and (S)-cEt ISIS 588856 Deoxy, MOE, 415 15 1.1 3.3 and (S)-cEt ISIS 588865 Deoxy, MOE, 362 14 2.0 3.3 and (S)-cEt ISIS 588867 Deoxy, MOE, 406 15 2.4 3.4 and (S)-cEt ISIS 588868 Deoxy, MOE, 399 15 1.5 3.4 and (S)-cEt ISIS 588870 Deoxy, MOE, 446 14 1.4 3.3 and (S)-cEt Study 6 (with MOE Gapmers, Deoxy, MOE and (S)-cEt Oligonucleotides, and (S)-cEt Gapmers)

Male rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers or with 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotide or (S)-cEt gapmer. One control group of 4 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L.

TABLE 195 Liver function markers Dose (mg/ ALT AST Albumin Chemistry kg/wk) (IU/L) (IU/L) (g/dL) PBS — — 54 73 4.3 ISIS 532770 5-10-5 MOE 100 57 114 4.4 ISIS 532800 5-10-5 MOE 100 176 180 4.3 ISIS 532809 5-10-5 MOE 100 71 132 4.1 ISIS 588540 5-10-5 MOE 100 89 202 4.4 ISIS 588544 5-10-5 MOE 100 75 152 3.9 ISIS 588548 5-10-5 MOE 100 50 71 4.1 ISIS 588550 5-10-5 MOE 100 80 133 3.6 ISIS 588553 5-10-5 MOE 100 59 112 3.9 ISIS 588555 5-10-5 MOE 100 97 142 3.8 ISIS 588848 Deoxy, MOE 50 53 82 3.9 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 50 198 172 4.4 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, urine levels of total protein and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 196 Total protein/creatinine ratio in the urine of rats Dose Chemistry (mg/kg/wk) P/C ratio PBS — — 1.1 ISIS 532770 5-10-5 MOE 100 8.3 ISIS 532800 5-10-5 MOE 100 6.5 ISIS 532809 5-10-5 MOE 100 6.1 ISIS 588540 5-10-5 MOE 100 10.1 ISIS 588544 5-10-5 MOE 100 7.9 ISIS 588548 5-10-5 MOE 100 6.6 ISIS 588550 5-10-5 MOE 100 7.6 ISIS 588553 5-10-5 MOE 100 7.0 ISIS 588555 5-10-5 MOE 100 6.2 ISIS 588848 Deoxy, MOE and (S)-cEt 50 5.2 ISIS 594430 3-10-3 (S)-cEt 50 5.3 Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. The results for the organ weights were expressed as a ratio to the body weights and normalized to the PBS control ratio.

TABLE 197 Organ weights/Body weight (BW) ratios Dose Spleen/ Liver/ Kidney/ Chemistry (mg/kg/wk) BW BW BW PBS — — 1.0 1.0 1.0 ISIS 532770 5-10-5 MOE 100 2.0 1.2 1.0 ISIS 532800 5-10-5 MOE 100 2.8 1.3 1.0 ISIS 532809 5-10-5 MOE 100 2.2 1.1 1.0 ISIS 588540 5-10-5 MOE 100 2.2 1.4 1.0 ISIS 588544 5-10-5 MOE 100 2.5 1.3 1.1 ISIS 588548 5-10-5 MOE 100 2.1 1.3 1.1 ISIS 588550 5-10-5 MOE 100 3.9 1.4 1.1 ISIS 588553 5-10-5 MOE 100 4.1 1.4 1.4 ISIS 588555 5-10-5 MOE 100 1.8 1.3 1.0 ISIS 588848 Deoxy, MOE 50 3.1 1.3 1.1 and (S)-cEt ISIS 594430 3-10-3 (S)-cEt 50 1.7 1.0 1.1

Example 128: Efficacy of Antisense Oligonucleotides Against CFB mRNA in hCFB Mice

Selected compounds were tested for efficacy in human CFB transgenic mice, founder line #6 The human CFB gene is located on chromosome 6: position 31913721-31919861. A Fosmid (ABC14-50933200C23) containing the CFB sequence was selected to make transgenic mice expressing the human CFB gene. Cla I (31926612) and Age I (31926815) restriction enzymes were used to generate a 22,127 bp fragment containing the CFB gene for pronuclear injection. DNA was confirmed by restriction enzyme analysis using Pvu I. The 22,127 bp DNA fragment was injected into C57BL/6NTac embryos. 6 positive founders were bred. Founder #6 expressed the liver human CFB mRNA and was crossbreed to the 3^(rd) generation. Progeny from 3^(rd) generation mice were used to evaluate human CFB ASOs for human CFB mRNA reduction.

Treatment

Groups of 3 mice each were injected subcutaneously twice a week for the first week with 50 mg/kg of ISIS oligonucleotides, followed by once a week dosing with 50 mg/kg of ISIS oligonucleotides for an additional three weeks. One control group of 4 mice was injected subcutaneously twice a week for 2 weeks for the first week with PBS for the first week for an additional three weeks. Forty eight hours after the last dose, mice were euthanized and organs and plasma were harvested for further analysis.

RNA Analysis

At the end of the dosing period, RNA was extracted from the liver and kidney for real-time PCR analysis of CFB mRNA levels. Human CFB mRNA levels were measured using the human primer probe set RTS3459. CFB mRNA levels were normalized to RIBOGREEN®, and also to the housekeeping gene, Cyclophilin. Results were calculated as percent inhibition of CFB mRNA expression compared to the control. All the antisense oligonucleotides effected inhibition of human CFB mRNA levels in the liver.

TABLE 198 Percent reduction of CFB mRNA levels in hCFB mice Normalized Normalized to to ISIS No RIBOGREEN Cyclophilin 532770 86 87 532800 88 87 532809 69 69 588540 95 94 588544 91 91 588548 78 77 588550 89 88 588553 94 94 588555 94 94 588848 83 82 594430 78 76

Example 129: In Vivo Antisense Inhibition of Murine CFB

Several antisense oligonucleotides were designed that were targeted to murine CFB mRNA (GENBANK Accession No. NM_008198.2, incorporated herein as SEQ ID NO: 5). The target start sites and sequences of each oligonucleotide are described in the table below. The chimeric antisense oligonucleotides in the table below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

TABLE 199 Gapmers targeting murine CFB Target Start Site on SEQ SEQ ID ISIS No Sequence ID NO: 5 NO 516269 GCATAAGAGGGTACCAGCTG 2593 804 516272 GTCCTTTAGCCAGGGCAGCA 2642 805 516323 TCCACCCATGTTGTGCAAGC 1568 806 516330 CCACACCATGCCACAGAGAC 1826 807 516341 TTCCGAGTCAGGCTCTTCCC 2308 808 Treatment

Groups of four C57BL/6 mice each were injected with 50 mg/kg of ISIS 516269, ISIS 516272, ISIS 516323, ISIS 516330, or ISIS 516341 administered weekly for 3 weeks. A control group of mice was injected with phosphate buffered saline (PBS) administered weekly for 3 weeks.

CFB RNA Analysis

At the end of the study, RNA was extracted from liver tissue for real-time PCR analysis of CFB, using primer probe set RTS3430 (forward sequence GGGCAAACAGCAATTTGTGA, designated herein as SEQ ID NO: 816; reverse sequence TGGCTACCCACCTTCCTTGT, designated herein as SEQ ID NO: 817; probe sequence CTGGATACTGTCCCAATCCCGGTATTCCX, designated herein as SEQ ID NO: 818). The mRNA levels were normalized using RIBOGREEN®. As shown in the Table below, some of the antisense oligonucleotides achieved reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 200 Percent inhibition of murine CFB mRNA in C57BL/6 mice ISIS No % 516269 29 516272 72 516323 77 516330 62 516341 72 Protein Analysis

CFB protein levels were measured in the kidney, liver, plasma, and in the eye by western Blot using goat anti-CFB antibody (Sigma Aldrich). Results are presented as percent inhibition of CFB, relative to PBS control. ‘n/a’ indicates that measurements were not taken for that sample. As shown in the Table below, antisense inhibition of CFB by ISIS oligonucleotides resulted in a reduction of CFB protein in various tissues. As shown in the Table below, systemic administration of ISIS oligonucleotides was effective in reducing CFB levels in the eye.

TABLE 201 Percent inhibition of murine CFB protein in C57BL/6 mice ISIS No Kidney Liver Plasma Eye 516269 20 58 n/a 70 516272 48 74 n/a 99 516323 73 85 90 93 516330 77 80 n/a n/a 516341 80 88 68 n/a

Example 130: Dose-Dependent Antisense Inhibition of Murine CFB

Groups of four C57BL/6 mice each were injected with 25 mg/kg, 50 mg/kg, or 100 mg/kg of ISIS 516272, and ISIS 516323 administered weekly for 6 weeks. Another two groups of mice were injected with 100 mg/kg of ISIS 516330 or ISIS 516341 administered weekly for 6 weeks. Two control groups of mice were injected with phosphate buffered saline (PBS) administered weekly for 6 weeks.

CFB RNA Analysis

RNA was extracted from liver and kidney tissues for real-time PCR analysis of CFB, using primer probe set RTS3430. The mRNA levels were normalized using RIBOGREEN®. As shown in the Table below, the antisense oligonucleotides achieved dose-dependent reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 202 Percent inhibition of murine CFB mRNA in C57BL/6 mice Dose ISIS No (mg/kg/wk) Liver Kidney 516272 25 39 32 50 73 36 100 87 42 516323 25 36 41 50 65 47 100 79 71 516330 100 85 45 516341 200 89 65 Protein Analysis

CFB protein levels were measured in the plasma by western Blot using goat anti-CFB antibody (Sigma Aldrich). As shown in the table below, antisense inhibition of CFB by the ISIS oligonucleotides resulted in a reduction of CFB protein. Results are presented as percent inhibition of CFB, relative to PBS control. ‘n/a’ indicates that measurements were not taken for that sample.

CFB protein levels were also measured in the eye by Western Blot. All treatment groups demonstrated an inhibition of CFB by 95%, with some sample measurements being below detection levels of the assay.

TABLE 203 Percent inhibition of murine CFB protein in C57BL/6 mice Dose ISIS No (mg/kg/wk) Liver 516272 25 32 50 70 100 83 516323 25 43 50 80 100 90 516330 100 n/a 516341 200 n/a

Example 131: Effect of Antisense Inhibition of CFB in the NZB/W F1 Mouse Model

The NZB/W F1 is the oldest classical model of lupus, where the mice develop severe lupus-like phenotypes comparable to that of lupus patients (Theofilopoulos, A. N. and Dixon, F. J. Advances in Immunology, vol. 37, pp. 269-390, 1985). These lupus-like phenotypes include lymphadenopathy, splenomegaly, elevated serum antinuclear autoantibodies (ANA) including anti-dsDNA IgG, a majority of which are IgG2a and IgG3, and immune complex-mediated glomerulonephritis (GN) that becomes apparent at 5-6 months of age, leading to kidney failure and death at 10-12 months of age.

Study 1

A study was conducted to demonstrate that treatment with antisense oligonucleotides targeting CFB would improve renal pathology in the mouse model. Female NZB/W F1 mice, 17 weeks old, were purchased from Jackson Laboratories. Groups of 16 mice each received doses of 100 μg/kg/week of ISIS 516272 or ISIS 516323 for 20 weeks. Another group of 16 mice received doses of 100 μg/kg/week of control oligonucleotide ISIS 141923 for 20 weeks. Another group of 10 mice received doses of PBS for 20 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. The mRNA levels were normalized using RIBOGREEN®. As shown in the Table below, some of the antisense oligonucleotides achieved reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 204 Percent inhibition of murine CFB mRNA in NZB/W F1 mice ISIS No Liver Kidney 516272 55 25 516323 63 43 141923 0 0 Proteinuria

Proteinuria is expected in 60% of animals in this mouse model. The cumulative incidence of severe proteinuria was measured by calculating the total protein to creatinine ratio using a clinical analyzer. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB achieved reduction of proteinuria in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 205 Percent cumulative incidence of severe proteinuria in NZB/W F1 mice % PBS 40 ISIS 516272 6 ISIS 516323 0 ISIS 141923 25 Survival

Survival of the mice was monitored by keeping count of the mice at the start of treatment and then again at week 20. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB increased survival in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 206 Number of surviving mice and % survival % survival at Week 1 Week 20 week 20 PBS 10 6 60 ISIS 516272 16 15 94 ISIS 516323 16 16 100 ISIS 141923 16 12 75 Glomerular Deposition

The amount of C3 deposition, as well as IgG deposition, in the glomeruli of the kidneys was measured by immunohistochemistry with an anti-C3 antibody. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB achieved reduction of both C3 and IgG depositions in the kidney glomeruli compared to the PBS control and the control oligonucleotide treated mice.

TABLE 207 Percent inhibition of glomerula deposition in NZB/W F1 mice ISIS No C3 IgG 516272 45 20 516323 48 2 141923 0 0 Study 2

Female NZB/W F1 mice, 16 weeks old, were purchased from Jackson Laboratories. A group of 10 mice received doses of 100 μg/kg/week of ISIS 516323 for 12 weeks. Another group of 10 mice received doses of 100 μg/kg/week of control oligonucleotide ISIS 141923 for 12 weeks. Another group of 10 mice received doses of PBS for 12 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the table below, treatment with ISIS 516323 achieved reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 208 Percent inhibition of murine CFB mRNA in NZB/W F1 mice ISIS No Liver Kidney 516323 75 46 141923 0 6 Proteinuria

The cumulative incidence of severe proteinuria was assessed by measuring urine total protein to creatinine ratio, as well as by measuring total microalbumin levels. The results are presented in the tables below and demonstrate that treatment with antisense oligonucleotides targeting CFB reduced proteinuria in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 209 Proteinuria in NZB/W F1 mice measured as urine microalbumin levels (mg/dl) ISIS No Week 0 Week 6 Week 8 Week 10 516323 0 0 5.4 0.4 141923 0 8.28 8.6 5.6

TABLE 210 Proteinuria in NZB/W F1 mice measured as total protein to creatinine ratio ISIS No Week 0 Week 6 Week 8 Week 10 516323 5.5 7.8 8.6 7.2 141923 6.9 10.0 13.5 7.2 Survival

Survival of the mice was monitored by keeping count of the mice at the start of treatment and then again at week 12. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB increased survival in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 211 Number of surviving mice Week 1 Week 12 PBS 10 9 ISIS 516323 10 10 ISIS 141923 10 9

Example 132: Effect of Antisense Inhibition of CFB in the MRL Mouse Model

The MRL/lpr lupus nephritis mouse model develops an SLE-like phenotype characterized by lymphadenopathy due to an accumulation of double negative (CD4⁻ CD8⁻) and B220⁺ T-cells. These mice display an accelerated mortality rate. In addition, the mice have high concentrations of circulating immunoglobulins, which included elevated levels of autoantibodies such as ANA, anti-ssDNA, anti-dsDNA, anti-Sm, and rheumatoid factors, resulting in large amounts of immune complexes (Andrews, B. et al., J. Exp. Med. 148: 1198-1215, 1978).

Treatment

A study was conducted to investigate whether treatment with antisense oligonucleotides targeting CFB would reverse renal pathology in the mouse model. Female MRL/lpr mice, 14 weeks old, were purchased from Jackson Laboratories. A group of 10 mice received doses of 50 μg/kg/week of ISIS 516323 for 7 weeks. Another group of 10 mice received doses of 50 jtg/kg/week of control oligonucleotide ISIS 141923 for 7 weeks. Another group of 10 mice received doses of PBS for 7 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the Table below, ISIS 516323 reduced CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 212 Percent inhibition of murine CFB mRNA in MRL/lpr mice ISIS No % 516323 68 141923 4 Renal Pathology

Renal pathology was evaluated by two methods. Histological sections of the kidney were stained with Haematoxylin & Eosin. The PBS control demonstrated presence of multiglomerular crescents tubular casts, which is a symptom of glomerulosclerosis. In contrast, the sections from mice treated with ISIS 516323 showed absent crescents tubular casts with minimal bowman capsule fibrotic changes, moderate to severe segmental mesangial cell expansion and glomerular basement membrane thickening.

Accumulation of C3 in the kidney was also assessed by immunohistochemistry with anti-C3 antibodies. The whole kidney C3 immunohistochemistry intensity score was calculated by intensity scoring system, which was computed by capturing 10 glomeruli per kidney and calculation the intensity of positive C3 staining. The results are presented in the table below and demonstrate that treatment with ISIS 516323 reduced renal C3 accumulation compared to the control groups.

TABLE 213 Renal C3 accumulation in MRL/lpr mice C3 quantification Whole kidney C3 (area/total area) % intensity score of average PBS PBS 2.5 100 ISIS 516323 1.6 68 ISIS 141923 2.2 99 Plasma C3 Levels

Reduction of CFB inhibits activation of the alternative complement pathway, preventing C3 consumption and leading to an apparent elevation of plasma C3 levels. Plasma C3 levels from terminal bleed were measured by clinical analyzer. The results are presented in the table below and demonstrate that treatment with ISIS 516323 increased C3 levels (p<0.001) in the plasma compared to the control groups.

TABLE 214 Plasma C3 levels (mg/dL) in MRL/lpr mice ISIS No. C3 516323 28 141923 16

The results indicate that treatment with antisense oligonucleotides targeting CFB reverses renal pathology in the lupus mouse model.

Example 133: Effect of Antisense Inhibition of CFB in the CFH Het Mouse Model

CFH heterozygous (CFH Het, CFH^(+/−)) mouse model express a mutant Factor H protein in combination with the full-length mouse protein (Pickering, M. C. et al., J. Exp. Med. 2007. 204: 1249-56). Renal histology remains normal in these mice up to six months old.

Study 1

Groups of 8 CFH^(+/−) mice, 6 weeks old, each received doses of 75 mg/kg/week of ISIS 516323 or ISIS 516341 for 6 weeks. Another group of 8 mice received doses of 75 mg/kg/week of control oligonucleotide ISIS 141923 for 6 weeks. Another group of 8 mice received doses of PBS for 6 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the Table below, the antisense oligonucleotides reduced CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 215 Percent inhibition of murine CFB mRNA in CFH^(+/−) mice ISIS No Liver Kidney 516323 80 38 516341 90 44 141923 0 17 Plasma C3 Levels

Reduction of CFB inhibits activation of the alternative complement pathway, preventing C3 consumption and leading to an apparent elevation of plasma C3 levels. Plasma C3 levels from terminal plasma collection were measured by clinical analyzer. The results are presented in the table below and demonstrate that treatment with ISIS 516323 increased C3 to normal levels in the plasma.

TABLE 216 Plasma C3 levels (mg/dL) in CFH^(+/−) mice ISIS No C3 516323 15 516341 17 141923 8 Study 2

Groups of 5 CFH^(+/−) mice each received doses of 12.5 mg/kg/week, 25 mg/kg/week, 50 mg/kg/week, 75 mg/kg/week, or 100 mg/kg/week of ISIS 516323 or ISIS 516341 for 6 weeks. Another group of 5 mice received doses of 75 μg/kg/week of control oligonucleotide ISIS 141923 for 6 weeks. Another group of 5 mice received doses of PBS for 6 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the Table below, the antisense oligonucleotides reduced CFB over the PBS control in a dose dependent manner. Results are presented as percent inhibition of CFB, relative to control.

TABLE 217 Percent inhibition of murine CFB mRNA in the liver of CFH^(+/−) mice Dose ISIS No (mg/kg/week) % 516323 12.5 34 25 51 50 72 75 79 100 92 516341 12.5 38 25 57 50 89 75 92 100 90 141923 75 13 Plasma C3 Levels

Reduction of CFB inhibits activation of the alternative complement pathway, preventing C3 consumption and leading to an apparent elevation of plasma C3 levels. Plasma C3 levels from terminal plasma collection were measured by clinical analyzer. The results are presented in the table below and demonstrate that treatment with ISIS oligonucleotides targeting CFB increased C3 levels in the plasma.

TABLE 218 Plasma C3 levels (mg/dL) in CFH^(+/−) mice Dose (mg/kg/week) C3 PBS — 10.1 516323 12.5 11.4 25 15.5 50 17.0 75 18.3 100 18.8 516341 12.5 12.1 25 16.3 50 18.6 75 22.1 100 19.1 141923 75 8.9

Example 134: Effect of ISIS Antisense Oligonucleotides Targeting Human CFB in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide efficacy and tolerability, as well as their pharmacokinetic profile in the liver and kidney, were evaluated.

At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (GENBANK Accession No. NW_001116486.1 truncated from nucleotides 536000 to 545000, designated herein as SEQ ID NO: 3). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide targeted to SEQ ID NO: 3 is presented in the Table below. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence. ‘Mismatches’ indicates the number of nucleobases in the human oligonucleotide that are mismatched with the rhesus genomic sequence.

TABLE 219 Antisense oligonucleotides complementary to the rhesus CFB genomic sequence (SEQ ID NO: 3) Target SEQ Start ID ISIS No Site Mismatches Chemistry NO 532770 6788 0 5-10-5 MOE 198 532800 7500 0 5-10-5 MOE 228 532809 7614 0 5-10-5 MOE 237 588540 7627 1 5-10-5 MOE 440 588544 7631 1 5-10-5 MOE 444 588548 7635 1 5-10-5 MOE 448 588550 7637 1 5-10-5 MOE 450 588553 7640 1 5-10-5 MOE 453 588555 7643 0 5-10-5 MOE 455 588848 7639 1 Deoxy, MOE and cEt 598 594430 6790 0 3-10-3 cEt 549 Treatment

Prior to the study, the monkeys were kept in quarantine for at least a 30 day period, during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 4 kg. Eleven groups of 4-6 randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS at four sites on the back in a clockwise rotation (i.e. left, top, right, and bottom), one site per dose. The monkeys were given four loading doses of PBS or 40 mg/kg of ISIS 532800, ISIS 532809, ISIS 588540, ISIS 588544, ISIS 588548, ISIS 588550, ISIS 588553, ISIS 588555, ISIS 588848, or ISIS 594430 for the first week (days 1, 3, 5, and 7), and were subsequently dosed once a week for 12 weeks (days 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84) with PBS or 40 mg/kg of ISIS oligonucleotide. ISIS 532770 was tested in a separate study with similar conditions with two male and two female cynomolgus monkeys in the group.

Hepatic Target Reduction

RNA Analysis

On day 86, liver and kidney samples were collected in duplicate (approximately 250 mg each) for CFB mRNA analysis. The samples were flash frozen in liquid nitrogen at necropsy within approximately 10 minutes of sacrifice.

RNA was extracted from liver and kidney for real-time PCR analysis of measurement of mRNA expression of CFB. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. RNA levels were also normalized with the house-keeping gene, Cyclophilin A. RNA levels were measured with the primer probe sets RTS3459, described above, or RTS4445_MGB (forward sequence CGAAGAAGCTCAGTGAAATCAA, designated herein as SEQ ID NO: 819; reverse sequence TGCCTGGAGGGCCCTCTT, designated herein as SEQ ID NO: 820; probe sequence AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815).

As shown in the Tables below, treatment with ISIS antisense oligonucleotides resulted in reduction of CFB mRNA in comparison to the PBS control. Analysis of CFB mRNA levels revealed that several of the ISIS oligonucleotides reduced CFB levels in liver and/or kidney. Here ‘0’ indicates that the expression levels were not inhibited. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions.

TABLE 220 Percent inhibition of CFB mRNA in the cynomolgus monkey liver relative to the PBS control RTS3459/ RTS445_(—) Cyclo- RTS3459/ MGB/Cyclo- RTS445_MGB/ ISIS No philin A RIBOGREEN philin A RIBOGREEN 532770* 12 37 24 45 532800 54 45 56 46 588540 31 27 28 24 588548 68 67 68 67 588550 53 39 51 37 588553 74 59 74 59 588555 73 71 71 69 588848 9 0 6 0 594430 24 26 23 25

TABLE 221 Percent inhibition of CFB mRNA in the cynomolgus monkey kidney relative to the PBS control RTS3459/ RTS445_(—) Cyclo- RTS3459/ MGB/Cyclo- RTS445_MGB/ ISIS No philin A RIBOGREEN philin A RIBOGREEN 532770* 34 56 2 31 532800 36 30 43 37 588540 70 71 67 69 588548 83 84 82 83 588550 81 77 78 74 588553 86 84 86 85 588555 32 34 48 50 588848 89 91 87 90 594430 33 37 19 23 Protein Analysis

Approximately 1 mL of blood was collected from all available animals at day 85 and placed in tubes containing the potassium salt of EDTA. The blood samples were placed in wet-ice or Kryorack immediately, and centrifuged (3000 rpm for 10 min at 4° C.) to obtain plasma (approximately 0.4 mL) within 60 minutes of collection. Plasma levels of CFB were measured in the plasma by radial immunodiffusion (RID), using a polyclonal anti-Factor B antibody. The results are presented in the Table below. ISIS 532770 was tested in a separate study and plasma protein levels were measured on day 91 or 92 in that group.

Analysis of plasma CFB revealed that several ISIS oligonucleotides reduced protein levels in a sustained manner. ISIS 532770, which was tested in a separate study, reduced CFB protein levels on day 91/92 by 50% compared to baseline values. The reduction in plasma CFB protein levels correlates well with liver CFB mRNA level reduction in the corresponding groups of animals.

TABLE 222 Plasma protein levels (% baseline values) in the cynomolgus monkey Day 1 Day 30 Day 58 Day 72 Day 86 PBS 113 115 95 83 86 ISIS 532800 117 68 52 39 34 ISIS 532809 104 121 100 80 71 ISIS 588540 108 72 61 40 38 ISIS 588544 118 74 53 33 29 ISIS 588548 110 41 28 20 16 ISIS 588550 104 64 54 38 37 ISIS 588553 97 42 35 18 16 ISIS 588555 107 35 37 18 18 ISIS 588848 116 95 92 69 71 ISIS 594430 104 64 59 45 46 Tolerability Studies Body Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured and are presented in the Table below. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides.

TABLE 223 Final body weights (g) in cynomolgus monkey Day 1 Day 14 Day 28 Day 42 Day 56 Day 70 Day 84 PBS 2887 2953 3028 3094 3125 3143 3193 ISIS 532770* 2963 2947 2966 3050 3097 3138 3160 ISIS 532800 2886 2976 3072 3149 3220 3269 3265 ISIS 532809 2755 2836 2927 2983 3019 3071 3098 ISIS 588540 2779 2834 2907 2934 2981 3034 3057 ISIS 588544 2837 2896 3009 3064 3132 3163 3199 ISIS 588548 2694 2816 2882 2990 3073 3149 3161 ISIS 588550 2855 2988 3062 3188 3219 3282 3323 ISIS 588553 3033 3156 3256 3335 3379 3372 3442 ISIS 588555 2757 2863 2965 3022 3075 3088 3158 ISIS 588848 2850 3018 3032 3187 3230 3212 3291 ISIS 594430 2884 2963 2953 3149 3187 3204 3256

TABLE 224 Final organ weights (g) in cynomolgus monkey Spleen Heart Kidney Liver PBS 2.8 11.6 11.9 55.8 ISIS 532770* 5.0 11.3 20.6 77.9 ISIS 532800 6.2 11.9 18.6 94.4 ISIS 588540 4.0 11.4 13.5 67.1 ISIS 588548 4.1 11.7 17.3 72.0 ISIS 588550 5.8 10.9 18.5 81.8 ISIS 588553 5.0 12.7 17.2 85.9 ISIS 588555 4.7 11.8 15.9 88.3 ISIS 588848 5.0 12.7 14.4 75.7 ISIS 594430 3.9 11.9 14.8 69.9 Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, blood samples were collected from all the study groups. The blood samples were collected from the cephalic, saphenous, or femoral veins, 48 hours post-dosing. The monkeys were fasted overnight prior to blood collection. Blood (1.5 mL) was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged (approximately 3,000 rpm for 10 min) to obtain serum. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).

Plasma levels of ALT and AST were measured and the results are presented in the Table below, expressed in IU/L. Bilirubin, a liver function marker, was similarly measured and is presented in the Table below expressed in mg/dL. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys. The results indicate that most of the antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides.

TABLE 225 Liver chemistry marker levels in cynomolgus monkey plasma on day 86 ALT AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 71 57 0.3 ISIS 532770* 59 58 0.1 ISIS 532800 65 86 0.1 ISIS 532809 35 58 0.1 ISIS 588540 70 88 0.2 ISIS 588544 55 97 0.2 ISIS 588548 61 85 0.2 ISIS 588550 94 84 0.2 ISIS 588553 44 65 0.2 ISIS 588555 63 84 0.2 ISIS 588848 69 65 0.2 ISIS 594430 86 53 0.2 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, blood samples were collected from all the study groups. The blood samples were collected from the cephalic, saphenous, or femoral veins, 48 hours post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged (approximately 3,000 rpm for 10 min) to obtain serum. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presented in the Table below, expressed in mg/dL. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys.

For urinalysis, fresh urine from all the animals was collected in the morning using a clean cage pan on wet ice. Food was removed overnight the day before urine collection but water was supplied. Urine samples (approximately 1 mL) were analyzed for protein to creatinine (P/C) ratio using a Toshiba 200FR NEO automated chemistry analyzer (Toshiba Co., Japan). ‘n.d.’ indicates that the urine protein level was under the detection limit of the analyzer.

The plasma and urine chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides.

TABLE 226 Renal chemistry marker levels (mg/dL) in cynomolgus monkey plasma on day 86 Total BUN Creatinine protein PBS 28 0.9 8.0 ISIS 532770* 20 0.9 6.9 ISIS 532800 25 0.9 7.5 ISIS 532809 23 0.8 7.4 ISIS 588540 30 0.8 7.5 ISIS 588544 26 0.9 7.4 ISIS 588548 25 0.9 7.6 ISIS 588550 24 0.9 7.2 ISIS 588553 25 0.8 7.2 ISIS 588555 25 0.8 7.6 ISIS 588848 24 0.9 7.5 ISIS 594430 25 0.8 7.2

TABLE 227 Renal chemistry marker levels in cynomolgus monkey urine on day 44 and day 86 Day 44 Day 86 PBS 0.03 n.d. ISIS 532800 0.01 n.d. ISIS 532809 0.01 n.d. ISIS 588540 0.03 n.d. ISIS 588544 0.01 0.09 ISIS 588548 0.01 0.01 ISIS 588550 0.04 0.01 ISIS 588553 0.05 n.d. ISIS 588555 0.03 0.03 ISIS 588848 0.09 n.d. ISIS 594430 0.03 n.d. Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 0.5 mL of blood was collected from each of the available study animals in tubes containing K₂-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer (Bayer, USA). The data is presented in the Tables below. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys.

The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose.

TABLE 228 Blood cell counts in cynomolgus monkeys RBC Platelets WBC Neutrophils Lymphocytes Monocytes (×10⁶/μL) (×10³/μL) (×10³/μL) (% WBC) (% total) (% total) PBS 5.8 347 9.4 42.7 53.1 3.0 ISIS 532770* 5.4 386 10.8 22.3 71.7 3.3 ISIS 532800 5.6 360 13.1 29.5 61.1 6.5 ISIS 532809 5.2 400 11.5 56.6 38.2 2.5 ISIS 588540 5.5 367 11.7 50.9 42.7 2.1 ISIS 588544 5.2 373 14.3 56.6 37.6 4.3 ISIS 588548 5.1 373 9.7 40.4 54.3 3.9 ISIS 588550 6.1 343 9.9 32.1 61.7 4.6 ISIS 588553 5.2 424 9.3 41.7 53.2 3.6 ISIS 588555 5.1 411 9.6 45.1 49.7 3.5 ISIS 588848 5.7 370 10.0 39.8 55.8 3.1 ISIS 594430 5.7 477 10.6 47.3 47.8 3.6

TABLE 229 Hematologic parameters in cynomolgus monkeys Hemoglobin HCT (g/dL) (%) PBS 14.1 46.6 ISIS 532770* 12.4 40.9 ISIS 532800 12.3 40.5 ISIS 532809 12.2 40.4 ISIS 588540 12.5 41.5 ISIS 588544 11.9 38.1 ISIS 588548 12.3 39.6 ISIS 588550 13.4 45.0 ISIS 588553 12.6 39.8 ISIS 588555 11.6 38.1 ISIS 588848 13.2 42.7 ISIS 594430 13.4 43.1 Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide was measured in the kidney and liver tissues. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in the Table below, expressed as μg/g liver or kidney tissue.

TABLE 230 Antisense oligonucleotide distribution Kidney Liver Kidney/Liver (μg/g) (μg/g) ratio ISIS 532800 3881 1633 2.4 ISIS 588540 3074 1410 2.2 ISIS 588548 3703 1233 3.0 ISIS 588550 4242 860 4.9 ISIS 588553 3096 736 4.2 ISIS 588555 4147 1860 2.2 ISIS 588848 2235 738 3.0 ISIS 594430 1548 752 2.1

Example 135: 6 Week Efficacy Study of Unconjugated and 5′-THA-GalNAc3 Conjugated Antisense Oligonucleotides Targeted to Human CFB in Transgenic Mice

Two antisense oligonucleotides having the same nucleobase sequence: uncongugated antisense oligonucleotide ISIS 588540 and 5′-THA-GalNAc3-conjugated antisense oligonucleotide ISIS 696844, were tested in human CFB transgenic mice (hCFB-Tg mice).

The mice were administered subcutaneously with ISIS 696844 at doses of 0.1, 1.25, 0.5, 2.0, 6.0, or 12.0 mg/kg/week or with ISIS 588540 at doses of 2, 6, 12, 25, or 50 mg/kg/week for 6 weeks. A control group of mice were administered subcutaneously with PBS for 6 weeks. Mice were sacrificed 48 hours after the last dose. Hepatic mRNA levels were analyzed by qRT-PCR.

Study 1

The results are presented in the Table below and demonstrate that the 5′-THA-GalNAc3-conjugated antisense oligonucleotide targeting CFB is more potent than the unconjugated antisense oligonucleotide with the same sequence.

TABLE 231 Efficacy of antisense oligonucleotides targeting CFB ED₅₀ ED₇₅ (mg/kg) (mg/kg) ISIS 588540 4.52 9.26 ISIS 696844 0.52 1.12 Study 2

Liver mRNA levels were measured with two different primer probe sets targeting different regions of the mRNA and normalized to either RIBOGREEN (RGB) or Cyclophilin. The primer probe sets were RTS3459, described above, and RTS3460 (forward sequence CGAAGCAGCTCAATGAAATCAA, designated herein as SEQ ID NO: 813; reverse sequence TGCCTGGAGGGCCTTCTT, designated herein as SEQ ID NO: 814; probe sequence AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815). The results are presented in the Table below and demonstrate that the 5′-THA-GalNAc3-conjugated antisense oligonucleotide targeting CFB is more potent than the unconjugated antisense oligonucleotide with the same sequence, irrespective of the primer probe set used.

TABLE 231 Efficacy of antisense oligonucleotides targeting CFB ED₅₀ ED₅₀ ED₅₀ ED₅₀ ED₇₅ ED₇₅ ED₇₅ ED₇₅ RTS3459 RTS3460 RTS3459 RTS3460 RTS3459 RTS3460 RTS3459 RTS3460 (RGB) (RGB) (Cyclophilin) (Cyclophilin) (RGB) (RGB) (Cyclophilin) (Cyclophilin) ISIS 588540 4.5 4.1 5.2 5.4 9.3 10.0 10.0 9.3 ISIS 696844 0.5 0.5 0.6 0.5 1.1 1.3 1.2 0.9 

What is claimed:
 1. A modified single-stranded oligonucleotide covalently attached to a conjugate group, wherein the modified single-stranded oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NO: 440, wherein the conjugate group covalently attached to the modified single-stranded oligonucleotide comprises:


2. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified oligonucleotide consists of 20 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO:
 440. 3. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified oligonucleotide consists of the nucleobase sequence of SEQ ID NO:
 440. 4. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising SEQ ID NO: 440, wherein the modified oligonucleotide comprises: a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; and a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
 5. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified oligonucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 440, wherein the modified oligonucleotide comprises a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; and a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.
 6. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified oligonucleotide consists of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 598, wherein the modified oligonucleotide comprises a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of three linked nucleosides; and a 3′ wing segment consisting of three linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment comprises a 2′-O-methoxyethyl sugar, 2′-O-methoxyethyl sugar, and cEt sugar in the 5′ to 3′ direction; wherein the 3′ wing segment comprises a cEt sugar, cEt sugar, and 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.
 7. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the oligonucleotide is at least 85% complementary to SEQ ID NO:
 1. 8. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar, or at least one modified nucleobase.
 9. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 8, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
 10. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 9, wherein the modified oligonucleotide comprises at least 1 phosphodiester internucleoside linkage.
 11. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 10, wherein each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.
 12. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 9, wherein each internucleoside linkage of the modified oligonucleotide comprises a phosphorothioate internucleoside linkage.
 13. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 8, wherein the modified sugar is a bicyclic sugar.
 14. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 13, wherein the bicyclic sugar is selected from the group consisting of: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)₂—O-2′ (ENA); and 4′-CH(CH₃)—O-2′ (cEt).
 15. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 8, wherein the modified sugar is 2′-O-methoxyethyl.
 16. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 8, wherein the modified nucleobase is a 5-methylcytosine.
 17. A modified double-stranded oligonucleotide, comprising the modified single stranded oligonucleotide covalently attached to a conjugate group of claim 1, and a second single stranded oligonucleotide hybridized to said modified single stranded oligonucleotide.
 18. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified single-stranded oligonucleotide comprises ribonucleotides.
 19. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the modified single-stranded oligonucleotide comprises deoxyribonucleotides.
 20. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide.
 21. The modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, wherein the conjugate group is linked to the modified oligonucleotide at the 3′ end of the modified oligonucleotide.
 22. A method of treating or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprising administering to the subject the modified single-stranded oligonucleotide covalently attached to a conjugate group of claim 1, thereby treating or ameliorating the disease.
 23. The method of claim 22, wherein the disease is macular degeneration, age related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy.
 24. The method of claim 22, wherein the disease is a kidney disease.
 25. The method of claim 24, wherein the kidney disease is lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS).
 26. A compound having the formula:

or a pharmaceutically acceptable salt thereof; wherein either R¹ is OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R² together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and R¹ and R² are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—; and for each pair of R³ and R⁴ on the same ring, independently for each ring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³ and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—; and R⁵ is selected from H and —CH₃; and Z is selected from S⁻ and O⁻.
 27. A compound having the formula:

or a pharmaceutically acceptable salt thereof.
 28. A compound having the formula:

or a pharmaceutically acceptable salt thereof.
 29. A compound having the formula:

or a pharmaceutically acceptable salt thereof.
 30. A compound having the formula:

or a pharmaceutically acceptable salt thereof.
 31. A composition comprising the compound of claim 27 or the pharmaceutically acceptable salt thereof.
 32. The compound of claim 27, wherein the pharmaceutically acceptable salt is sodium.
 33. A method of treating or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprising administering to the subject the compound of claim 27, or pharmaceutically acceptable salt thereof, thereby treating or ameliorating the disease.
 34. The method of claim 33, wherein the disease is macular degeneration, age related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy.
 35. The method of claim 33, wherein the disease is a kidney disease.
 36. The method of claim 35, wherein the kidney disease is lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS). 